Lessells, C.M.; Mateman, A.C.
We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird spec
Laboratory-scale composts in which oily sludge was composted under mesophilic conditions with amendments such as peat, bark, and fresh or decomposed horse manure, were studied with respect to basic parameters such as oil degradation, respirometry, and bacterial numbers. Further, an attempt was made to characterize a part of the bacterial flora using randomly amplified polymorphic DNA (RAPD). The compost based on decomposed horse manure showed the greatest reduction of oil (85%). Comparison with a killed control indicated that microbial degradation actually had occurred. However, a substantial part of the oil was stabilized rather than totally broken down. Volatiles, on the contrary, accounted for a rather small percentage (5%) of the observed reduction. RAPD indicated that a selection had taken place and that the dominating microbial flora during the active degradation of oil were not the same as the ones dominating the different basic materials. The stabilized compost, on the other hand, had bacterial flora with similarities to the ones found in peat and bark
Nielsen, Karen L; Godfrey, Paul A; Stegger, Marc;
Identifying and characterizing clonal diversity are important when analysing fecal flora. We evaluated random amplified polymorphic DNA (RAPD) PCR, applied for selection of Escherichia coli isolates, by whole genome sequencing. RAPD was fast, and reproducible as screening method for selection...
Bansal, N S; McDonell, F
The randomly amplified polymorphic DNA (RAPD) technique was used in the development of a fingerprinting (typing) and identification protocol for Legionella strains. Twenty decamer random oligonucleotide primers were screened for their discriminatory abilities. Two candidate primers were selected. By using a combination of these primers, RAPD analysis allowed for the differentiation between all different species, between the serogroups, and further differentiation between subtypes of the same ...
Han, Feng-chan; Ng, Han-Chong; Ho, Bow
AIM: H pylori genomes are highly diversified. This project was designed to genotype H pylori isolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing.
Tegelström, H; Höggren, M
We performed breeding experiments with adders (Vipera berus) to determine whether multiple matings may result in multiple paternity. DNA fingerprinting of mothers, their offspring, and possible fathers using a polydinucleotide probe [(TG)n] gave a low overall similarity between unrelated individuals (0.18 +/- 0.07; SD) and an average of 17 bands that were male-specific. In no cases were there fewer than seven paternal-specific bands present in the fingerprint of an offspring, enabling us unambiguously to identify the biological father among five males. Multiple paternity was detected in the investigated broods with offspring sired exclusively by the captive males. PCR amplification of random amplified polymorphic DNA (RAPD) using 16 decamer primers gave 76 bands and an average similarity of 0.95 (+/- 0.01) between the males, which were collected at different, geographically well-separated localities. Although there were on average 8.3 (+/- 1.9) bands that differ between males in pairwise comparisons, there were only 1.9 (+/- 1.1) bands per male that are specific for a particular individual. Thus, RAPDs are adequate for paternity determination only in experiments with a low number of males, whereas DNA fingerprinting offers sufficient information to discriminate between large numbers of putative fathers. PMID:7826312
Corney, B G; Colley, J; Djordjevic, S P; Whittington, R.; Graham, G C
We compared random amplified polymorphic DNA (RAPD) fingerprinting with cross-absorption agglutination and restriction enzyme analysis for typing bovine leptospires. Using RAPD fingerprinting, we examined a number of Leptospira serovars, namely, hardjo genotypes bovis and prajitno, pomona, balcanica, tarassovi, swajizak, kremastos, australis, and zanoni, which are likely to be isolated from Australian cattle. Each serovar and genotype had a unique RAPD profile. Of 26 field isolates of Leptosp...
Winget, Danielle M.; Wommack, K Eric
Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation o...
Random amplified polymorphic DNA (RAPD) markers were successfully used in discrimination of sexes in Nile tilapia fish ( Oreochromis niloticus) using linear discriminant function analysis. The results provide support for the view that major genetical sex determining factors exist in tilapia.
Halima Hassan Salem; Bahy Ahmed Ali; Tian-Hua Huang; Da-Nian Qin; Xiao-Mei Wang; Qing-Dong Xie
The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA (RAPD) technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation.Also we discuss the advantages and limitations of RAPD.Molecular markers have entered the scene of genetic improvement in different fields of agricultural research.The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.
Twenty four wheat varieties/lines were assessed through RAPD for genetic diversity. Of forty primers, thirteen were able to amplify the genomic DNA and yielded 269 polymorphic bands. The percentage of the polymorphic loci was 86.22%. Nei's genetic diversity (h) ranged from 0.248 to 0.393, with an average of 0.330. Shanon's index ranged from 0.382 to 0.567, with an average of 0.487. The proportion of genetic variation among the populations ( Ds) accounted for 28.58 % of the whole genetic diversity. The level of gene flow (Nm) was 1.25. Some specific RAPD bands were also identified, variety C-591, and QM-4531 contain a specific segment of 4.9 kbp. Whereas SARC-1 and PKV-1600 amplified a specific DNA segment with primer A-09. Marvi-2000 contains two specific segments of 3.2 kb and 200 bp amplified with primer B-07. Genetically most similar genotypes were C-591 and Pasban-90 (76%) and most dissimilar genotypes were Rawal-87 and Khirman (36.1%). On the basis of results, 24 wheat varieties under study could be divided into 'two' groups and five clusters 'A' to 'E. (author)
Iñiguez Alena M
Full Text Available The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.
Sithithaworn, Paiboon; Nuchjungreed, Chadaporn; Srisawangwong, Tuanchai; Ando, Katsuhiko; Petney, Trevor N.; Chilton, Neil B.; Andrews, Ross H.
Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People’s Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these...
Wilkerson, R C; Parsons, T J; Albright, D G; Klein, T A; Braun, M J
The usefulness of random amplified polymorphic DNA (RAPD) was examined as a potential tool to differentiate cryptic mosquito species. It proved to be a quick, effective means of finding genetic markers to separate two laboratory populations of morphologically indistinguishable African malaria vectors, Anopheles gambiae and An. arabiensis. In an initial screening of fifty-seven RAPD primers, 377 bands were produced, 295 of which differed between the two species. Based on criteria of interpretability, simplicity and reproducibility, thirteen primers were chosen for further screening using DNA from thirty individuals of each species. Seven primers produced diagnostic bands, five of which are described here. Some problematic characteristics of RAPD banding patterns are discussed and approaches to overcome these are suggested. PMID:8269099
Shioda, Hiroko; Satoh, Kanako; Nagai, Fumiko; Okubo, Tomoko; Seto, Takako; Hamano, Tomoko; Kamimura, Hisashi; Kano, Itsu
Juice and integument of leaves of 3 Aloe species, Aloe vera, A. ferox and A. africana, are not allowed to be used as food according to the Pharmaceutical Affairs Law in Japan. On the other hand, whole leaves of A. arborescens can be used as food. The present study was designed to distinguish Aloe species by random amplified polymorphic DNA (RAPD) analysis. DNA was isolated from fresh and dried leaves of the 4 Aloe species. Five out of 32 different 10-mer primers examined were useful for analysis. By comparison of the characteristic bands of PCR products on agarose gel, it was possible to distinguish the 4 species. Thus, the botanical species of Aloe in commercial food products can be identified by RAPD analysis. PMID:14606430
Carmen Elisa Posso
Full Text Available Random amplified polymorphic DNA (RAPD markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8 but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8. According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001. The variation among individuals within populations (phiST 0.8869, P < 0.001was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.
Patrícia M Pinto
Full Text Available The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV. These samples were subjected to randomly amplified polymorphic DNA (RAPD analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.
Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R
The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level. PMID:26782491
The increasing presence of genotoxic pollutants in the equatic environment has led to the development of quick monitoring methods.We have applied the random amplified polymorphism DNA(RKAPD) method to evaluate the toxic effects of genotoxic chemicals.In all 22 of normal wild type Danio rerio(zebrafish),78 amplified bands were obtained after PCR using primers set,in which the frequency of emergency above 60 percent is 21.There do exist 4 stable bands in the amplifed product,which appear in all normal test samples.The zebrafish RAPD fingerprinting database is set up on this base.The RAPD pattern from zebrafishes exposed to genotoxic chemicals such as cyclophosphamide 0.1-10mg·L-1 and dimethoate 10-100mg·L-1 displayed some changes in polymorphism of band pattern including the lost of stable bands.There also exists distinct distance between the band pattern of exposed zebrafishes and the control samples via the cluster method.In addition,the result derived from numeric analysis is more sensitive than the result obtained from stable band analysis because it can reveal the distance between the band pattern of 0.05mg·L-1 cyclophosphamide exposed zebrafish and the control sample.These results showed the accuracy and the sensitivity of the two analyses of genotoxicity based on the RAPD fingerprinting research.
Budi Setiadi Daryono
Full Text Available A random amplified polymorphic DNA (RAPD marker linked to powdery mildew resistance gene (Pm-I in melon PI 371795 was reported. However, the RAPD marker has problem in scoring. To detect powdery mildew resistance gene (Pm-I in melon accurately, the RAPD marker was cloned and sequenced to design sequence characterized amplified region (SCAR markers. SCAPMAR5 marker derived from pUBC411 primer yielded a single DNA band at 1061 bp. Segregation of SCAPMAR5 marker in bulk of F2 plants demonstrated that the marker was co-segregated with RAPD marker from which the SCAR marker was originated. Moreover, results of SCAR analysis in diverse melons showed SCAPMAR5 primers obtained a single 1061 bp linked to Pm-I in resistant melon PI 371795 and PMAR5. On the other hand, SCAPMAR5 failed to detect Pm-I in susceptible melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to Pm-I evaluated by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers. Key words: Cucumis melo L., Pm-I, RAPD, SCAPMAR5
Full Text Available Olive (Olea europea L. is one of the oldest cultivated plants characteristic in the Mediterranean area, where it is the most important oilproducing crop. The cultivated olive (O. europaea L. var. europaea is propagated by cutting or grafting, whereas wild olive (O. europaea L. var. sylvestris is reproduced from seeds. These two olive types are interfertile and have led to a large number of varieties. Morphological descriptions are not entirely reliable, due to numerous synonyms and homonyms in designations, labelling mistakes, the presence of varietal clones, and the uncertain identification methods thus far applied. Molecular markers, as random amplified polymorphic DNA (RAPD markers, are environment-independent and efficient to identify olive varieties and to detect synonymous and homonymous. In this study, fifteen selected RAPD markers are used for determination of relationships among twenty individuals belonging to four important Turkish olive cultivars. Our results showed that RAPD markers can be used to differentiate olive cultivars
Ping-Ping Yang, Rong-De Ma1, Xue Zhao and Rui-Liang Zhu*
Full Text Available To study the similarity among Bordetella avium isolates in China, antigens and diagnostic antiserum of 22 B. avium isolates were prepared for serotyping, and a set of 20 commercially available primers was screened out to identify suitable primers for random amplified polymorphic DNA fingerprinting (RAPD analysis in this study. Twenty-two B. avium isolates were divided into two serovars (A and B based on their reaction in the plate-agglutination test. Four primers R1, R2, R4 and R10 resulted in informative fingerprints and were used to evaluate the B. avium isolates. Based on their RAPD patterns, a dendrogram allowed the separation of the B. avium isolates into six genetic similarity clusters. However, no direct correlation was observed between serotypes and RAPD typing among the isolates.
Grayson, T H; Atienzar, F A; Alexander, S M; Cooper, L F; Gilpin, M L
The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture. PMID:10618262
Zhi-Hong Xian; Wen-Ming Cong; Shu-Hui Zhang; Meng-Chao Wu
AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments.METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD)with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated,purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data.RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size,histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene.CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcinogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis.
Winget, Danielle M; Wommack, K Eric
Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information. PMID:18344351
Full Text Available Qicai Liu1,2, Ailin Liu3,4, Feng Gao5, Shaohuang Weng3,4, Guangxian Zhong3,4, Jingfeng Liu6, Xinhua Lin3,4, Jian-hua Lin7, Xuhai Chen81Department of Laboratory Medicine, Fujian Medical University, Fuzhou, 2Department of Gene Diagnosis, Fujian Medical University, Fuzhou, 3Department of Nano-Biomedical Research Center, Fujian Medical University, Fuzhou, 4Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou, 5Department of Pathology, Fujian Medical University, Fuzhou, 6Department of Surgery, Fujian Medical University, Fuzhou, 7Department of Bone Oncology, First Affiliated Hospital, Fujian Medical University, Fuzhou, 8College of Electrical Engineering and Automation, Fuzhou University, Fuzhou, People's Republic of ChinaObjective: To review the feasibility of coupling the techniques of random amplified polymorphic DNA (RAPD with carbon nanotube-based modified electrode for guanine/deoxy-guanine triphosphate (dGTP electrochemical sensing for mapping of the pancreatic cancer genetic fingerprint and screening of genetic alterations.Methods: We developed a new method to study the electrochemical behavior of dGTP utilizing carbon multiwalled nanotube (MWNT-modified glassy carbon electrodes (GCEs. RAPD was applied for amplification of DNA samples from healthy controls and patients with pancreatic cancer under the same conditions to determine the different surplus quantity of dGTP in the polymerase chain reaction (PCR, thereby determining the difference/quantity of PCR products or template strands. Using this method we generated a genetic fingerprint map of pancreatic cancer through the combination of electrochemical sensors and gel electrophoresis to screen for genetic alterations. Cloning and sequencing were then performed to verify these gene alterations.Results: dGTP showed favorable electrochemical behavior on the MWNTs/GCE. The results indicated that the electrical signal and dGTP had a satisfactory linear relationship with the d
Larissa Brandão Góes
Full Text Available Random Amplified Polymorphic DNA (RAPD procedure was used to examine the genetic variability among fourteen isolates of Trichoderma and their ability to antagonize Rhizoctonia solani using a dual-culture assay for correlation among RAPD products and their hardness to R. solani. Seven oligodeoxynucleotide primers were selected for the RAPD assays which resulted in 197 bands for 14 isolates of Trichoderma. The data were entered into a binary matrix and a similarity matrix was constructed using DICE similarity (SD index. A UPGMA cluster based on SD values was generated using NTSYS (Numerical Taxonomy System, Applied Biostatistics computer program. A mean coefficient of similarity obtained for pairwise comparisons among the most antagonics isolates was around 40%. The results presented here showed that the variability among the isolates of Trichoderma was very high. No relationship was found between the polymorphism showed by the isolates and their hardness, origin and substrata.A técnica de RAPD (Random Amplified Polymorphic DNA foi utilizada para examinar a variabilidade genética em quatorze isolados de Trichoderma além de sua capacidade de antagonizar o fungo fitopatogênico Rhizoctonia solani usando pareamento in vitro, e a possível relação entre perfís de RAPD e agressividade dos isolados de Trichoderma a R. solani. Foram selecionados sete primers para os ensaios de RAPD, os quais produziram 197 bandas. Os dados foram introduzidos no programa de computador NTSYS (Numerical Taxonomy System, Applied Biostatisticsna forma de uma matrix binária, sendo construída uma matriz de similaridade utilizando-se o coeficiente de similaridade de DICE (SD e baseado nos valores SD, pelo método de agrupamento UPGMA um dendrograma. Observou-se que o grau de similaridade das amostras que apresentaram melhor desempenho antagônico foi bastante baixo, em torno de 40%. Os resultados demonstraram que a variabilidade entre os isolados de Trichoderma é muito
Wong, Kwong-Kwok; Tsang, Yvonne T.M.; Shen, Jianhe; Cheng, Rita S.; Chang, Yi-Mieng; Man, Tsz-Kwong; Lau, Ching C.
Besides their use in mRNA expression profiling, oligonucleotide microarrays have also been applied to single-nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) or allelic imbalance studies. In this report, we evaluate the reliability of using whole genome amplified DNA for analysis with an oligonucleotide microarray containing 11 560 SNPs to detect allelic imbalance and chromosomal copy number abnormalities. Whole genome SNP analyses were performed with DNA extracted from osteosar...
Daffonchio, Daniele; Borin, Sara; Frova, Giuseppe; Gallo, Romina; Mori, Elena; Fani, Renato; Sorlini, Claudia
Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a pu...
Antolin, M F; Bosio, C. F.; COTTON, J; W. Sweeney; Strand, M.R.; Black-IV, W. C.
The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a d...
Baurand, Pierre-Emmanuel; Capelli, Nicolas; de Vaufleury, Annette
The study explores the relevance of coupling Random Amplified Polymorphic DNA (RAPD) and a High-Resolution capillary electrophoresis System (HRS) method for assessing the genotoxic potential of the wide variety commercial formulations of pesticides. Using this technique, the genotoxic potential of a glyphosate-based herbicide (Roundup Flash(®) (RU)) and two fungicide formulations based on tebuconazole and copper (Corail(®) and Bordeaux mixture (BM), respectively) was evaluated on terrestrial snail embryos. Clutches of Cantareus aspersus were exposed during their entire embryonic development to a range of concentration around the EC50 values (based on hatching success) for each compound tested. Three primers were used for the RAPD amplifications of pesticides samples. RAPD-HRS revealed concentration-dependent modifications in profiles generated with the three primers in RU(®)-exposed embryos from 30 mg/L glyphosate. For Corail(®)-exposed embryos, only two of the three primers were able to show alterations in profiles from 0.05 mg/L tebuconazole. For BM-exposed embryos, no signs of genotoxicity were observed. All changes observed in amplification profiles have been detected at concentrations lower than the recommended doses for vineyard field applications. Our study demonstrates the efficiency of coupling RAPD and HRS to efficiently screen the effect of pesticide formulations on DNA. PMID:26160746
Full Text Available Amorphophallus muelleri Blume (Araceae is valued for its glucomanan content for use in food industry (healthy diet food, paper industry, pharmacy and cosmetics. The species is triploid (2n=3x=39 and the seed is developed apomictically. The present research is aimed to identify genetic variability of six population of A. muelleri from Java (consisted of 50 accessions using random amplified polymorphic DNA (RAPD. The six populations of the species are: East Java: (1 Silo-Jember, (2 Saradan-Madiun, (3 IPB (cultivated, from Saradan-Madiun, (4 Panti-Jember, (5 Probolinggo; and Central Java: (6 Cilacap. The results showed that five RAPD primers generated 42 scorable bands of which 29 (69.05% were polymorphic. Size of the bands varied from 300bp to 1.5kbp. The 50 accessions of A. muelleri were divided into two main clusters, some of them were grouped based on their populations, and some others were not. The range of individual genetic dissimilarity was from 0.02 to 0.36. The results showed that among six populations investigated, Saradan population showed the highest levels of genetic variation with mean values of na = 1.500+ 0.5061, ne = 1.3174 + 0.3841, PLP = 50% and He = 0, 0.1832+0.2054, whereas Silo-Jember population showed the lowest levels of genetic variation with mean values na = 1.2619+ 0.4450, ne = 1.1890 + 0.3507, PLP = 26.19% and He = 0.1048+0.1887. Efforts to conserve, domesticate, cultivate and improve genetically should be based on the genetic properties of each population and individual within population, especially Saradan population which has the highest levels of genetic variation, need more attention for its conservation.
Full Text Available Eurycoma longifolia Jack is one of the extensively exploited medicinal plants in Indonesia. The objectives of this study were to obtain information on genetic diversity and population genetic structure of E. longifolia to formulate effective conservation plan. RAPD marker was used to assess the genetic diversity of E. longifolia collected from 5 natural populations in Riau Province. A total of 25 plants were analyzed using 5 RAPD primers, which amplified produced 44 scored DNA bands. The mean observed number of alleles per locus (No, number of effective alleles (Ne, and percentage of polymorphic loci (PPL of E. longifolia were 1.57, 1.34, and 56.80%, respectively. The degree of differentiation among populations of E. longifolia was 0.31 (Ht = 0.29; Hs = 0.20. The mean value of estimated gene flow among populations of E. longifolia was 1.11 individual per generation. The UPGMA dendogram formed 2 significant clusters. The first cluster consisted of Pelalawan and Kampar populations, while the second cluster was formed from Kuansing, Rohul, and Rohil population. The genetic diversity information in this study is very important to perform efficient conservation and effective future management of its genetic resources.Keywords: Eurycoma longifolia, RAPD marker, genetic variation, conservation DOI: 10.7226/jtfm.19.2.138
Nogueira, C A; Stafuzza, N B; Ribeiro, T P; Prado, A D L; Menezes, I P P; Peixoto, N; Gonçalves, P J; Almeida, L M
Hancornia speciosa, popularly known as mangabeira, is a fruit tree native to the Brazilian Cerrado that shows great economic potential, due to its multiple uses. Intraspecific classification of this species is difficult because it shows high morphological diversity. An early study of the species reported that there are six botanic varieties that differ morphologically mainly in the shapes of their leaves and flowers. Except to note the wide morphological variation and economic potential of this species, few studies have been published about the genetic diversity of mangabeira. Knowledge of the genetic variability of this species among populations would be useful for genetic conservation and breeding programs. Therefore, we tested the transferability of 12 simple sequence repeats from expressed sequence tags (EST-SSRs) from Catharanthus roseus to H. speciosa and used 10 random amplified polymorphic DNA markers to evaluate the genetic variability among botanical varieties of H. speciosa. We obtained a high transferability frequency of EST-SSR markers from C. roseus to H. speciosa (75%). However, EST-SSR markers showed low heterozygosity and locus variability (two or three alleles by locus), which suggest low genetic diversity in the mangabeira samples. The Jaccard dissimilarity index and an examination of geographic distances indicated a non-spatial structuring of the genetic variability. Our markers were unable to distinguish H. speciosa botanical varieties. PMID:26662392
Hua YANG; Si-Ming GAN; Guang-Tian YIN; Huang-Can XU
The random amplified polymorphic DNA (RAPD) molecular marker technique was used to determine the sex of Calamus simplicifolius C. F. Wei In the present study, DNA samples were extracted individually from 10 male and 10 female plants. After a total of 1 040 decamer primers had been tested, an approximate 500-bp male-specific DNA fragment was generated with the S1443 primer. It is feasible to identify sex at the early stages of plant life, which is beneficial for improving breeding programs of this dioecious species. In addition, we have obtained a proper RAPD protocol that is useful for other species of rattan.
Zehr Emilie S; Lavrov Dennis V; Tabatabai Louisa B
Abstract Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for dis...
Shamsuddeen Rufai; Hanafi, M. M.; Rafii, M. Y.; Ahmad, S; Arolu, I. W.; Jannatul Ferdous
The knowledge of genetic diversity of tree crop is very important for breeding and improvement program for the purpose of improving the yield and quality of its produce. Genetic diversity study and analysis of genetic relationship among 20 Moringa oleifera were carried out with the aid of twelve primers from, random amplified polymorphic DNA marker. The seeds of twenty M. oleifera genotypes from various origins were collected and germinated and raised in nursery before transplanting to the fi...
Eribe, Emenike Ribs K.; Olsen, Ingar
In a polyphasic approach to improve the taxonomy of Leptotrichia we have recently demonstrated heterogeneity in the enzymatic/biochemical reactions and cellular fatty acid content of Leptotrichia isolates. In the present study, SDS-PAGE of whole-cell proteins and random amplified polymorphic DNA (RAPD) analyses were used to further characterize and distinguish these isolates of which 57 had been assigned as Leptotrichia buccalis and two as ‘Leptotrichia pseudobuccalis’. The phylog...
Kermani, Firoozeh; Shams-Ghahfarokhi, Masoomeh; Gholami-Shabani, Mohammadhassan; Razzaghi-Abyaneh, Mehdi
In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method. PMID:27116962
DING Wei; ZHANG Zhengwang; CHANG Jiang; ZHANG Er; WU Xiushan; ZHANG Jinguo
The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifica tions showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.
Saulnier, P; Bourneix, C; Prévost, G; Andremont, A
Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best metho...
Ciğerci, İbrahim Hakkı; Cenkci, Süleyman; Kargıoğlu, Mustafa; Konuk, Muhsin
This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC50 and 2×EC50 values were 8 and 16 mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10 mg/l) and 8 and 16 mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96 h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique. PMID:25550040
High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis
Vogel, Birte Fonnesbech; Fussing, V.; Ojeniyi, B.; Gram, Lone; Ahrens, Peter
other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was...... different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for...... virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains...
Jong-Man Yoon; Gye-Woong Kim
Genetic similarity and diversity of cultured catfish Silurus asotus populations collected from two areas in western Korea were examined using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Out of 20 random primers tested, 5 produced 1344 RAPD bands ranging from 8.2 to 13.6 polymorphic bands per primer. The polymorphic bands in these populations ranged from 56.4% to 59.6%. Polymorphic bands per lane within populations ranged from 4.9% to 5.3%. The similarity within the Kunsan population varied from 0.39 to 0.82 with a mean (± SD) of 0.56 ± 0.08. The level of bandsharing values was 0.59 ± 0.07 within the catfish population from Yesan. The genetic similarity in cultured catfish populations may have been caused because individuals from two populations were reared in the same environmental conditions or by inbreeding during several generations. However, in view of bandsharing values, polymorphic bands and also the specific major bands that were inter-population-specific, significant genetic differentiation between these populations were present even if bandsharing (BS) values were somewhat numerically different. Therefore, the number of RAPD polymorphisms identified in this study may be sufficient to permit estimating genetic similarity and diversity. However, in future, additional populations, sampling sites and individuals will be necessary to make up for these weak points.
F. Robert; Lebreton, F; Bougnoux, M. E.; Paugam, A; Wassermann, D.; Schlotterer, M; Tourte-Schaefer, C; Dupouy-Camet, J
Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hôpital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. a...
Neveu, H; Hafen, T; Zimmermann, E; Rumpler, Y
Continued survival of most animal species depends on population management and active protection. It is generally agreed that, in order to avoid extinction of endangered species, ex situ and in situ conservation must be developed in tandem. However, even though many recommendations have been put forward to promote the survival of captive populations, some rapidly become extinct due to loss of genetic diversity (drift effect). Genetic markers, such as random amplified polymorphic DNA (RAPD) markers, can be applied to rapid testing of many individuals. They also permit analysis of very small amounts of DNA, when small species such as mouse lemurs (Microcebus) are to be tested. Using RAPD markers, we compare genetic diversity in four captive groups of Microcebus murinus to that in a sample of 70 wild mouse lemurs. Following the principles of Mendelian inheritance, each amplified fragment of DNA may be considered as a 'locus' (or an amplifying site). The series of bands amplified by a particular primer in any individual is referred to as the individual's 'profile'. We tested 5 primers, or, in the above terms, we studied 98 different 'loci'. Results showed that the captive groups had lost genetic information with respect to the wild sample. Among the four captive groups, the loss of genetic diversity varied according to their number of founders and/or the management of their captive reproduction. Our study of polymorphism permitted us to establish tools for the genetic management of captive breeding, and for the determination of paternity which frequently give better results than behavioural studies; and simulation of introductions or departures of individuals in one very monomorphic group permitted estimation of future increases in its genetic diversity. PMID:9595690
Abdolaziz Rastegar Lari; Bagher Yakhchali; Parviz Owlia; Hassan Salimi
One of the major opportunistic pathogens in patients with burninjuries is Pseudomonas aeruginosa, which causes severe infectionsin burned patients. The objective of the study was to examinethe molecular epidemiology of P. aeruginosa colonization inthe burn unit of Shahid Motahari Hospital in Tehran, Iran. Restrictionfragment length polymorphism (RFLP) and random amplifiedpolymorphic DNA (RAPD) analysis were employed tostudy 127 clinical and two environmental P. aeruginosa isolatescollected fr...
Full Text Available The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed.
Robert, F; Lebreton, F; Bougnoux, M E; Paugam, A; Wassermann, D; Schlotterer, M; Tourte-Schaefer, C; Dupouy-Camet, J
Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hôpital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. albicans strains isolated from various anatomical sites of unrelated patients, and the strains showed 22 different patterns. Random amplified polymorphic DNA was then used in the epidemiological surveillance of the patients in the burn unit over a 9-month period. Seven patterns were identified among 84 isolates from 18 patients. One pattern (pattern A) corresponding to isolates from 7 of the 18 patients (68% of isolates) predominated throughout the 9-month study, while some strains with other profiles were isolated only once. Some profiles appeared to show a particular geographic pattern within the unit, suggesting transmission from room to room. These results underline the importance of fungal surveillance in such patients and the need to inform nursing staff of measures to prevent the spread of Candida spp. from patient to patient. PMID:7494029
Use of random amplified polymorphic DNA (RAPD to detect DNA damage induced by Prangos ferulacea (Umbelliferae essential oil against the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae
Ercan Fahriye Sumer
Full Text Available DNA damage detection in Ephestia kuehniella by RAPD-PCR Abstract - The random amplified polymorphic DNA (RAPD assay is a useful method for detecting genotoxin-induced DNA damage. In the present study, this assay was evaluated to measure the essential oil of Prangos ferulacea-induced DNA changes in Ephestia kuehniella Zeller larvae. The third instar larvae of E. kuehniella were exposed to 500 μl L-1 essential oil for 24 h. Forty random primers were used for RAPD-PCR of which eleven produced unique polymorphic band patterns. In comparison with control larvae, the essential oil-treated larvae showed greater changes in RAPD profiles. This is the first report of an analysis of the genotoxic effect of P. ferulacea essential oil against E. kuehniella larvae using RAPD-PCR.
Wensheng Wang; Xiuqin Zhao; Yajiao Pan; Linghua Zhu; Binying Fu; Zhikang Li
DNA methylation,one of the most important epigenetic phenomena,plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli.In the present study,a rnethylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress.Consistent with visibly different phenotypes in response to salt stress,epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salttolerant FL478 and salt-sensitive IR29.In addition,most tissue-specific DNA methylation loci were conserved,while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes.Strikingly,salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478.This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage,and helps to further elucidate the implications of DNA methylation in crop growth and development.
KUSUMADEWI SRI YULITA
Full Text Available Yulita KS (2011 Genetic variations of Lansium domesticum Corr. accessions from Java, Bengkulu and Ceram based on Random Amplified Polymorphic DNA fingerprints. Biodiversitas 12: 125-130. Duku (Lansium domesticum Corr. is one of popular tropical fruits in SE Asia. The spesies has three varieties, known as duku, langsat and kokosan; and duku is the most popular one for being the sweetiest fruit. Indonesia has several local varieties of duku, such as duku Condet, duku Sumber and duku Palembang. This present study aimed to assess genetic diversity of 47 accessions of duku from Java, Sumatra, and Ceram based on RAPD fingerprints. Ten RAPD’s primers were initially screened and five were selected for the analysis. These five primers (OPA 7, 13, 18, OPB 7, and OPN 12 generated 53 scorable bands with an average of 10.6 polymorphic fragment per primer. Percentage of polymorphism ranged from 16.89% (OPA 7 and OPN 12 to 24.54% (OPB 7 with an average of 20.16% polymorphism. OPB 7 at 450 bp was exclusively possessed by accession 20 (Java, OPA 18 at 500 bp was by accession 6 (Java, 550 bp by 3 clones from Bengkulu. While OPN 12 at 300 bp and OPA 13 at 450 bp were shared among the accessions. Clustering analysis was performed based on RAPD profiles using the UPGMA method. The range of genetic similarity value among accessions was 0.02-0.65 suggesting high variation of gene pool existed among accessions.
Uso do Random Amplified Polymorphic DNA (RAPD no estudo populacional do Triatoma brasiliensis Neiva, 1911 Use of Random Amplified Polymorphic DNA (RAPD in the populational study of Triatoma brasiliensis Neiva, 1911
Érika C. Borges
Full Text Available Para o estudo de variabilidade genética em Triatoma brasiliensis, o principal vetor da doença de Chagas no Nordeste brasileiro, espécimes de três diferentes populações intradomiciliares foram analisados. Regiões do DNA genômico foram amplificadas utilizando dois iniciadores randômicos através da técnica de RAPD (Random Amplified Polymorphic DNA, visualizados em géis de poliacrilamida corados pela prata. Os perfis originados se mostraram bastante homogêneos quando comparados intrapopulacionalmente. Populações capturadas em duas regiões diferentes do Estado do Ceará também apresentaram homogeneidade entre si, mas, quando comparadas com a população proveniente do Piauí, foi possível diferenciá-las. Esses resultados, preliminares, indicam que o RAPD pode ser usado com sucesso nos estudos de variabilidade em triatomíneos, bem como sugerem a existência de variabilidade entre diferentes populações de T. brasiliensis pertencentes a uma mesma subespécie.We evaluated the genetic variability of Triatoma brasiliensis, the main vector of Chagas disease in Northeast Brazil, using specimens from three populations. Regions of genomic DNA were amplified by RAPD (Random Amplified Polimorphic DNA, using two primers. The products were visualized after polyacrylamide gel electrophoresis followed by silver staining. A dendrogram constructed through the Dice similarity coefficient allowed for separation of the tested specimens into three distinct groups. The populations captured in areas from Ceará State showed similar profiles, but different from that captured in Piauí State. Our results indicate that RAPD can be used successfully in triatomine studies and suggest the presence of genetic variability between different populations of T. brasiliensis.
Sharma, A K; Mendki, M J; Tikar, S N; Chandel, K; Sukumaran, D; Parashar, B D; Veer, Vijay; Agarwal, O P; Prakash, Shri
Genetic variability and environmental factors may influence the refractiveness, propagation of pathogen and transmission of disease. Random amplified polymorphic DNA (RAPD) is one of the widely used molecular markers for population genetic diversity studies. In present study, RAPD is used to ascertain the genetic variability in Culex quinquefasciatus populations collected from various Indian geographical locations. Out of 50 RAPD primers screened, 14 primers exhibited clear, concrete and distinct banding pattern showing up to 100% polymorphism. Primer OPBD3 was tested with DNA of 14 geographical populations from India (including one laboratory population) showed 21 loci representing 14 populations with 100% polymorphism. The genetic diversity among the populations indicated the Shannon index (I) and gene diversity index (H(ST)), 0.48 and 0.31, respectively among the population, displaying rich genetic variation among the Cx. quinquefasciatus populations. Consensus tree showed two clusters indicating the genetic variation among the various geographical populations. The findings of this study may be useful to understand the population variation under different ecological conditions and development of effective vector management strategies. PMID:19577531
Muhammad Ayub Khan; Malik Ashiq Rabbani; Muharnmad Munir; Saifullah Khan Ajmal; Muhammad Azim Malik
Genetic diversity among 45 Indian mustard (Brassica Juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession 'Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.
Saulnier, P; Bourneix, C; Prévost, G; Andremont, A
Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best method of typing methicillin-resistant S. aureus strains. PMID:8463406
Full Text Available Random amplified polymorphic DNA (RAPD analysis was performed on 38 cultivars of cherry (Prunus avium L. grown in the Jerte Valley, Cáceres, Spain. Thirty five selected decamer primers produced 69 reproducible polymorphic amplification products. The degree of polymorphism detected made possible the identification of all the cultivars by combining the RAPD banding patterns of only seven primers: OPK-08, OPQ-14, OPR-09, OPS-19, OPX-02, OPX-15 and OPZ-13. Eleven unique markers allowed identification of nine cultivars while 15 cultivars were identified by unique banding patterns. A similarity matrix derived from the RAPD amplification products generated by all the primers was obtained using the index of similarity of Jaccard. The similarity coefficients among cultivars ranged from 0.27 to 0.81 with an average of 0.50. A dendrogram based on UPGMA clustering method was constructed using the similarity matrix. The dendrogram showed a good correlation between the clustering of cherry cultivars and their geographic origin, especially revealing a stronger genetic proximity between some of the most characteristic cultivars of the Jerte Valley. This result supports the autochthonous origin hypothesis for these cultivars.
Tulsiani, Suhella; Craig, S B; Graham, G C;
High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as ...
Abdolaziz Rastegar Lari
Full Text Available One of the major opportunistic pathogens in patients with burninjuries is Pseudomonas aeruginosa, which causes severe infectionsin burned patients. The objective of the study was to examinethe molecular epidemiology of P. aeruginosa colonization inthe burn unit of Shahid Motahari Hospital in Tehran, Iran. Restrictionfragment length polymorphism (RFLP and random amplifiedpolymorphic DNA (RAPD analysis were employed tostudy 127 clinical and two environmental P. aeruginosa isolatescollected from January to June 2008. In RFLP, the PCR productsof 16S rRNA gene were digested with restriction enzyme Alu I,Hae III, and Rsa I, and the fragments generated were analyzed byagarose electrophoresis. Molecular typing by RFLP did show nodiscriminatory power for P. aeruginosa isolates, but RAPD-PCRrevealed eight different genotypes; RAPD1to RAPD8 in clinicaland environmental isolates. RAPD1 was the major genotype inclinical (n=64, 50.4% and environmental isolates (n=1, 50%.The findings suggest that RAPD might have a superior typeabilityand discriminatory power over RFLP to study P. aeruginusa.Moreover, they highlight the need for further attention to the controlof infection sources in Burn Units to prevent the transmissionof the bacterium.
Comfort O. AFOLAYAN
Full Text Available The genetic differentiation of Archachatina marginata populations from three different zones of Nigeria was studied with a view to delimiting them into sub-species. One hundred and nineteen (119 snail specimens were collected, comprising of forty (40 specimens from Yenagoa (Mangrove forest and from Kabba (Guinea Savanna and thirty nine (39 specimens were from Ile-Ife (Rainforest. Eight parameters of the shell specimens of A. marginata which included height of shell, width of shell, aperture height, aperture width, spire length, spire width, penultimate whorl length and first whorl length were subjected to Principal Component Analysis (PCA and Canonical Variates Analysis (CVA to delimit the populations into sub-species. DNA of the various populations was extracted from the foot muscle using CTAB (Cetyl Trimethyl Ammonium Bromide method, which was subjected to RAPD analysis. The RAPD studies employed five (5 oligonucleotide primers (OPB – 17, OPH – 12, OPH – 17, OPI – 06 and OPU – 14 to amplify DNA from 27 samples of A. marginata selected. All five primers produced different band patterns, and the number of fragments amplified per primer varied. Among them, OPB- 17 gave DNA profiles with more numerous bands than the others primers. Both PCA and CVA produced overlapped clusters of A. marginata specimens from the three vegetation zones. The height of shell was observed to be the most variable feature and preferably the most suitable parameter for population grouping. Analysis of the proportions of polymorphic loci and band sharing based on similarity indices for A. marginata samples indicated a relatively high level of genetic variation in the populations from the three areas.
LI Ling-hua; HU Feng-yu; CHEN Wan-shan; CAI Wei-ping; SONG Wei-nan; KUANG Yan-ling; TANG Xiao-ping
Background Penicillium mameffei (P.mameffei) is an emerging pathogenic fungus that can cause invasive mycosis in patients with AIDS.The epidemiological features of P.mameffei infection in AIDS patients in Guangdong province remain unclear so far.This study aimed to investigate the genetic diversity within a population of 163 P.mameffei isolates obtained from AIDS patients and search for the dominant clinical strains in Guangdong province.Methods One hundred and sixty-three P.marneffeiisolates obtained from AIDS patients in Guangdong province during January 2004 and December 2009 were studied by randomly amplified polymorphic DNA (RAPD) using two random primers (H2 and H22).The degree of similarity between samples was calculated through similarity coefficients from RAPD fragment data and the dendrogram was assessed using the unweighted pair group method with arithmetic mean (UPGMA).Results Two primers showed a high degree of discrimination and good stability.Primer H2 yielded eight different patterns (H2-1 to H2-8) among 163 isolates with the discriminatory power being 0.413.Primer H22 identified seven types (H22-1 to H22-7) among 163 isolates with the discriminatory power being 0.467.Genetic similarity coefficients based on RAPD data among 163 P.marneffei isolates ranged from 0.681 to 0.957,61.96％ of which were no less than 0.83.The discriminatory power of the two primers was 0.524.One hundred and sixty-three P.marneffei isolates were clustered into nine distinct groups (groups I to IX) at the similarity coefficient value of 0.83 and group Ⅰ was the most common,including 101 strains (61.96％).Conclusion The RAPD analyses could provide important information as to the degree of genetic diversity and the relationship among clinical P.mameffei isolates,revealing genetic polymorphism and dominant strains.
Vogel, Birte Fonnesbech; Jørgensen, Lasse Vigel; Ojeniyi, B.;
One hundred and forty-eight Listeria monocytogenes isolates originating from vacuum packed cold-smoked salmon produced in 10 different Danish smokehouses were compared by Random Amplified Polymorphic DNA (RAPD) profiling. A total of 16 different reproducible RAPD profiles were obtained using a...... indicate a possible persistence of closely related strains of L. monocytogenes in smokehouses. (C) 2001 Elsevier Science B.V. All rights reserved...
Vogel, Birte Fonnesbech; Jørgensen, Lasse Vigel; Ojeniyi, B.; Huss, Hans Henrik; Gram, Lone
One hundred and forty-eight Listeria monocytogenes isolates originating from vacuum packed cold-smoked salmon produced in 10 different Danish smokehouses were compared by Random Amplified Polymorphic DNA (RAPD) profiling. A total of 16 different reproducible RAPD profiles were obtained using a st...... indicate a possible persistence of closely related strains of L. monocytogenes in smokehouses. (C) 2001 Elsevier Science B.V. All rights reserved...
Asensio, Luis; González, Isabel; Fernández, Alicia; Rodríguez, Miguel A; Lobo, Esther; Hernández, Pablo E; García, Teresa; Martín, Rosario
A random amplified polymorphic DNA (RAPD) method was developed for the specific identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. Using two different reaction primers (S1 and L1), RAPD analysis produced clear fingerprints from which the three fish species could be easily identified. This approach is rapid and reliable and offers the potential to detect fraudulent or unintentional mislabeling of these species in routine seafood authentication analysis. PMID:11848581
YUYU SURYASARI POERBA
Full Text Available Dyera costulata (Miq. Hook.f (Apocynaceae is a large tree of the lowland tropical rain forest of Southeast Asia, Thailand, Malay Peninsula and Indonesia, especially in Sumatra and Kalimantan (Borneo islands. Its economic value was in its copious latex, used as gum in the manufacture of chewing gum. Today the timber of this species is much sought after for the manufacture of pencils and picture frames. Information on genetic diversity of the species is very limited. Hence studies were initiated to screen primers for RAPD analyses of Dyera costulata for use in genetic variation studies. Seventy one Operon primers (10 mer were used to generate a total of 864 consistent and ambiguous amplification products ranging from 200 bp to 2.0 kb. Rare and genotype specific bands were identified which could be effectively used to distinguish the genotypes. 34 highly polymorphic primers (100% are recorded from 71 primers used. Three primers (OPA-04, OPU-06, and OPU-07 produced highest variable RAPD profiles. The dendrogram separated the 8 genotypes into 2 groups. Genetic dissimilarity ranged from 0.07 to 0.71 %.
Evânia Galvão Mendonça; Anderson Marcos de Souza; Fábio de Almeida Vieira; Regiane Abjaud Estopa; Cristiane Aparecida Fioravante Reis; Dulcinéia de Carvalho
The objective of this study was to assess the genetic variability in two natural populations of Calophyllum brasiliense located along two different rivers in the state of Minas Gerais, Brazil, using RAPD molecular markers. Eighty-two polymorphic fragments were amplified using 27 primers. The values obtained for Shannon index (I) were 0.513 and 0.530 for the populations located on the margins of the Rio Grande and Rio das Mortes, respectively, demonstrating the high genetic diversity in the st...
Atienzar, Franck A; Jha, Awadhesh N
The random amplified polymorphic DNA (RAPD) is a useful assay for the detection of genotoxin-induced DNA damage and mutations. In this study, we have further evaluated the potential of this assay to measure benzo(a)pyrene [B(a)P]-induced DNA changes, and repair (in kinetic experiments) as well as transgenerational effects in the water fleas, Daphnia magna. The organisms, which reproduce parthenogenetically, were exposed to 50 microg L(-1) B(a)P for 3 or 6 days and were allowed to recover in clean medium for 12 or 9 days, respectively. Qualitative and quantitative changes were observed in RAPD profiles generated not only from the B(a)P exposed Daphnia but also from previously treated organisms during the recovery experiments. The fact that some of the RAPD changes disappeared at the end of both recovery experiments suggested that the DNA effects were fully repaired or reversed. In addition, some of the B(a)P-induced RAPD alterations detected in parental D. magna were also observed in the offspring patterns. This suggested that DNA alterations that occurred in germ cells were probably transmitted to the next cohorts. The present study shows that the RAPD method can be useful to qualitatively assess the kinetics of DNA changes, repair and transgenerational effects and such effects could potentially be linked to survival and reproductive success at higher levels of biological organisation. In addition, the water fleas have efficient capabilities to repair or reverse B(a)P-induced DNA effects. Finally, unrepaired or misrepaired genetic damage induced by genotoxins such as B(a)P could be transmitted to next generations in these parthenogenetically reproducing organisms. PMID:15288546
Iñiguez Alena M; Araújo Adauto; Ferreira Luiz Fernando; Vicente Ana Carolina P
The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pre...
Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng
DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. PMID:25461675
Full Text Available As an aim in sustainable agriculture, biological control of plant diseases has received intensive attention mainly as a response to public concern about the use of chemical fungicides in the environment. Soil Actinomycetes particularly Streptomyces spp. enhance soil fertility and have antagonistic activity against wide range of plant pathogens. To investigate for biocontrol means against the pathogen, 30 isolates of Actinomycetes have been isolated from agricultural soils of Kerman province of Iran and assayed for antagonistic activity against Botrytis allii, the agent of onion gray mold. RAPD DNA analysis has been used to determine the relatedness of active and non-active isolates based on their RAPD-PCR fingerprints. PCR amplifiable DNA samples have been isolated using the CTAB method and amplified fragments have been obtained from 5 random 10-mer primers. Different DNA fingerprinting patterns have been obtained for all of the isolates. Electrophoretic and cluster analysis of the amplification products has revealed incidence of polymorphism among the isolates. A total of 138 bands, ranging in size from 150-2800 bp, have been amplified from primers which 63.7% of the observed bands have been polymorphic. Genetic distances among different varieties have been analyzed with a UPGMA (Unweighted pair-group method, arithmetic average-derived dendrogram. Resulting dendrogram has showed from 0.65 to 0.91 similarities among varieties and divided the isolates into five major groups. Isolates which haven’t had any antagonistic activity against B. allii have been separated into a group and other isolates classified into four groups. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.
Full Text Available The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment Length Polymorphisms (AFLP technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1 Kaeg Dum and Malador (2 Kaeg Nuan (3 Pakchong and Solo (4 Taiwan (5 Co Coa Hai Nan and (6 Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found.
Ratchadaporn, Janthasri; Sureeporn, Katengam; Khumcha, U
The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment Length Polymorphisms (AFLP) technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top) were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC) of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1) Kaeg Dum and Malador (2) Kaeg Nuan (3) Pakchong and Solo (4) Taiwan (5) Co Coa Hai Nan and (6) Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found. PMID:19090101
Siti Azizah Mohd Nor; Abu Talib A; Mohd Ghows M A; Samsudin B
In a preliminary investigation, Random Amplified Polymorphie DNA (RAPD) analysis and partial mitochon-drial ND2 gene sequencing were conducted to study the genetic variation of the Indian mackerel, Rastrelliger kanagurta along a 450 km stretch of its distribution on the west coast of Peninsular Malaysia. A total of 53 individuals from 6 popu-lations were analyzed using 4 RAPD primers and a sub-sample of 15 individuals was chosen for sequencing of partial ND2 gene. Comparison between the 2 markers revealed genetic structuring in the RAPD results but genetic homogeneity for ND2 gene. Based on the former there may be at least 2 genetically differentiated groups of Rastrelliger kanagurta a-long this stretch.
Identification of a differentially expressed thymidine kinase gene related to tapping panel dryness syndrome in the rubber tree (Hevea brasiliensis Muell. Arg. by random amplified polymorphic DNA screening
Full Text Available Tapping panel dryness (TPD syndrome is one of the latex yield affecting factors in the rubber tree (Hevea brasiliensis Mull. Arg.. Therefore, identification of a DNA marker will be highly useful for screening progenies in breeding programs. The major goal of this study was to detect genetic variations and/or identification of gene fragments among 37 Hevea clones by the random amplified polymorphic DNA “fingerprinting” technique. Different levels of DNA polymorphism were detected with various primers and a distinct polymorphic band (2.0 kb was obtained with OPA-17 primer. It was cloned into a plasmid vector for further sequence characterization and the nucleotide sequence shows homology with a novel putative plant thymidine kinase (TK gene, designated as HbTK (Hevea brasiliensis thymidine kinase; GenBank accession number AY130829. The protein HbTK has 67%, 65%, 64%, and 63% similarity to TK genes of Medicago, Oryza, Arabidopsis, and Lyco- persicon, respectively, and it was highly conserved in all species analyzed. The predicted amino acid sequence contained conserved domains of TK proteins in the C-terminal half. Southern blot analysis indicated that HbTK is one of the members of a small gene family. Northern blot results revealed that the expression of the HbTK gene was up-regulated in mature bark tissues of the healthy tree while it was down-regulated in the TPD-affected one. These results suggest that this gene may play important roles in maintaining active nucleotide metabolism during cell division at the tapped site of bark tissues in the healthy tree under stress (tapping conditions for normal latex biosynthesis.
Full Text Available Background. The number of Helicobacter pylori clones infecting a single host has been discussed in numerous reports. The number has been suggested to vary depending on the regions in the world. Aim. The purpose of this study was to examine the number of clones infecting a single host in a Japanese urban population. Materials and Methods. Thirty-one Japanese patients undergoing upper gastrointestinal endoscopy were enrolled in this study. H. pylori isolates (total 104 strains were obtained from biopsy specimens (antrum, corpus, and duodenum and gastric juice. Clonal diversity was examined by the random amplified polymorphic DNA (RAPD fingerprinting method. Results. The RAPD fingerprinting patterns of isolates from each patient were identical or very similar. And the isolates obtained from several patients with 5- to 9-year intervals showed identical or very similar RAPD patterns. Conclusion. Each Japanese individual of an urban population is predominantly infected with a single H. pylori clone.
Bello Martha Darce
Full Text Available A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis. The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%, C. tropicalis (23%, C. glabrata (14% and C. krusei (4%. This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.
Magariños, B; Toranzo, A E; Barja, J L; Romalde, J L
In this work, we applied the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida), an important pathogen for different marine fish. Regardless of the oligonucleotide primer employed, the 29 isolates of Ph. damselae subsp. piscicida tested were separated into two groups, the RAPD-PCR analysis differentiated the European strains from the Japanese strains. The similarity between both groups estimated on the basis of the Dice coefficient was 75-80%. These results show that European and Japanese isolates of Ph. damselae subsp. piscicida, regardless of their host fish species, belong to two different clonal lineages. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen. PMID:11057980
Pamela E. Akin-Idowu
Full Text Available Efficient utilization of plant genetic resources for nutrition and crop improvement requires systematic understanding of the important traits. Amaranthus species are distributed worldwide with an interesting diversity of landraces and cultivars whose leaves and seeds are consumed. Despite their potential to enhance food security and economic livelihoods, grain amaranth breeding to improve nutritional quality and adoption by farmers in sub-Saharan Africa is scanty. This study assessed the variation among 29 grain amaranth accessions using 27 phenotypic (10 morphological and 17 nutritional characters and 16 random amplified polymorphic DNA (RAPD primers. Multivariate analysis of phenotypic characters showed the first four principal components contributing 57.53% of observed variability, while cluster analysis yielded five groups at 87.5% similarity coefficient. RAPD primers generated a total of 193 amplicons with an average of 12.06 amplicons per primer, 81% of which were polymorphic. Genetic similarities based on Jaccard’s coefficient ranged from 0.61 to 0.88. The RAPD-based unweighted pair group method with arithmetic mean dendrogram grouped the accessions into nine clusters, with the same species clustering together. RAPD primers distinguished the accessions more effectively than phenotypic markers. Accessions in the different clusters as obtained can be exploited for heterotic gain in desired nutritional traits.
Robarts, Daniel W. H.; Wolfe, Andrea D.
In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open readin...
@@ 家蚕微粒子病是由微粒子原虫(微孢子虫)类寄生于蚕体引起的病害.微粒子病对蚕丝业生产危害极大,一直被列为蚕种制造检疫对象.许多学者对病原的传播途径、侵染机制及分类地位作了大量研究.其中微孢子虫的分类又以血清学关系、发育增殖方式和孢子超微结构作为主要划分依据,这些表型依据仍然具有局限性.随机扩增多态性DNA技术(Randomly Amplified Polymorphic DNA,RAPD)因能方便、快速提供DNA多态性的丰富信息而被广泛用于动植物和微生物的遗传差异、亲缘关系和进化分类等领域,但应用于微孢子虫的分类研究尚未见报道.本文用60个随机引物构建了8种微孢子虫的DNA指纹图谱,以探讨其相互间的亲缘关系.
Full Text Available A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV, Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP method. Transcript derived fragments (TDFs identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future.
Lin, Yu-Tsung; Jan, Fuh-Jyh; Lin, Chia-Wei; Chung, Chien-Hung; Chen, Jo-Chu; Yeh, Shy-Dong; Ku, Hsin-Mei
A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future. PMID:23874746
Puccinia hordei is the causal agent of barley leaf rust. To study the genetic diversity in P. hordei, 45 isolates with diverse virulence patterns and geographical origins were analyzed using amplified fragment length polymorphism markers. Two pathotypes of Puccinia graminis f. sp. tritici and one is...
Random Amplified Polymorphic DNA (RAPDs) markers were utilized to detect polymorphism between pure lines and commercially available Basmati rice varieties to assess variation which may be helpful in quality control and varietal identification (Basmati-370 and derived radiation induced mutants), differentiation of mutants and parents, and identification of RAPD markers co-segregating with important agronomic traits including plant height, days to flower and grain quality. Basmati varieties were distinguished from non-Basmati varieties with the help of five diagnostic markers which will be useful for detecting mixing of non-Basmati and Basmati rices, currently a serious marketing problem. Different Basmati cultivars were identified with the help of diagnostic RAPD markers which can be used in quality control as well as for ''fingerprinting'' of cultivars. Different radiation induced mutants were also successfully distinguished from the parents on the basis of variety specific and mutant specific markers which will be useful for varietal identification. In addition to this, other markers were also identified which can differentiate mutants from each other and are being, used for the fingerprinting of different mutants, particularly the dwarf mutants having similar appearance but different parentage. For identification of RAPD markers co-segregating with plant height and days to flower, 50 F2 plants and four F3 families were studied from a reciprocal cross made between Kashmir Basmati (tall and early) and Basmati-198 (dwarf and late). Segregating bands were observed within these populations, and indicating the possible use of RAPD markers for tagging gene(s) of agronomic importance in rice. (author)
Random amplified polymorphic DNA markers for DNA fingerprinting and genetic variability assessment of minute parasitic wasp species (Hymenoptera: Mymaridae and Trichogrammatidae) used in biological control programs of phytophagous insects.
Landry, B S; Dextraze, L; Boivin, G
Biological control of insects that feed on our crops has become more practical in recent years by mass release of egg parasitoid microhymenoptera. Trichogramma species are now commercially reared and spread in commercial fields to control specific insect pests. Microhymenoptera species are, however, very small and morphologically indistinguishable within species, although strains of a given species differ in their efficiency to control specific insect pests. Traditional taxonomy is unable to differentiate microhymenoptera species at the strain level. It is becoming increasingly important to develop a reliable system to monitor genetic variations both within and between strains of commercially important microhymenoptera, to detect genetic drift occurring during several generations of multiplication, to protect patents, and to certify the lots of commercially released microhymenoptera. We have developed a system based on DNA markers to rapidly characterize individuals of five species of microhymenoptera from the genus Anaphes and Trichogramma including a new species of Anaphes not previously described. The main components of our system are a rapid and simple DNA micro-extraction method and fast DNA polymorphism analyses based on random amplified polymorphic DNA markers. PMID:8349128
Tulsiani, Suhella; Craig, S B; Graham, G C;
A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to d...
Waldron Julie; Peace Cameron P.; Searle Iain R.; Furtado Agnelo; Wade Nick; Findlay Ian; Graham Michael W.; Carroll Bernard J.
Arbitrarily-primed DNA markers can be very useful for genetic fingerprinting and for facilitating positional cloning of genes. This class of technologies is particularly important for less studied species, for which genome sequence information is generally not known. The technologies include Randomly Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting (DAF), and Amplified Fragment Length Polymorphism (AFLP). We have modified the DAF protocol to produce a robust PCR-based DNA ma...
Biernasiuk, Anna; Korona-Głowniak, Izabela; Grzegorczyk, Agnieszka; Malm, Anna
Cancer patients are predisposed to fungal infections caused by Candida albicans, especially to oral or respiratory tract candidiasis. The aim of this study was to estimate genetic diversity by RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) of C. albicans isolated from upper respiratory tract of 100 patients with non-small cell lung cancer. Among 52 strains, 34 genotypes were defined. 10 clusters comprising 28 (53.85%) isolates with similarity coefficient ≥ 80% were formed. The remaining 24 (46.15%) isolates represented individual genotypes. The RAPD-PCR technique revealed genomic variability within C. albicans isolated from upper respiratory tract of the cancer patients. PMID:25371918
"Use of Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR and ITS2 PCR assays for differentiation of populations and putative sibling species of Anopheles fluviatilis (Diptera: Culicidae in Iran"
SR Naddaf Dezfouli
Full Text Available Anopheles fluviatilis complex is known to be a vector of malaria in Iran. Since mosquitoes of this species cover a wide geographical range in Iran, they might have evolved into different separated populations. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR assay was used to differentiate geographic populations of this species. DNA was extracted from individual mosquitoes from 8 localities in 4 south and southeast provinces and amplified in PCR reactions using 18 single primers of arbitrary nucleotide sequence. Results of RAPD-PCR showed that Kazeroun populations could simply be differentiated from other populations using a diagnostic fragment amplified with primer UBC-306. But other populations could not be differentiated either visually or by means of statistical analysis. Moreover ITS2 fragments of some selected specimens were amplified using a pair of universal primer and sequenced as a key standard for detection of putative sibling species. Sequence analysis of the ITS2 fragments revealed a very high (100% homology among the populations. These findings are crucial in epidemiological studies concerning relatedness of geographic populations and vector movement in the region. Results of RAPD-PCR and ITS2 analysis suggest that this taxon in Iran comprises of only one species with a low genetic variation among geographic populations.
This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...
Kokotovic, Branko; Friis, N.F.; Jensen, J.S.; Ahrens, Peter
Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including...... Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneunmoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5), AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and Mfe...... discriminate the analyzed strains at species and intraspecies levels as well, Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis, Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma...
Khush, R S; Becker, E; Wach, M.
Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spo...
蒋建平; 杨懋勋; 黄永芳
representative mountain areas in Guangdong China were analyzed by RAPD markers , so as to provide good foundation for further natural germplasm resources and related studies of Lilium brownii populations, and provide good evidence of the discrimination of Brown lily and Greenish lily at molecular level. DNA from all samples was extracted from leaves and scales, which amplified clear, bright and steady RAPD bands. 20 primers were screened from 273 random primers, and a total 433 DNA bands were amplified, 430 (99. 3% ) of which were polymorphic, the percentage of polymorphic bands (PPB) at the population level was 60. 67% on average. The unweighted pair group method arithmetic average (UPGMA) clustery analysis of the RAPD data from 199 samples showed that all Brown lily samples and Greenish lily samples in the same population separated obviously atthe value of 0. 68 of genetic similarity coefficient, which was a good evidence of the discrimination of Brown lily and Greenish lily at molecular level.
郭安平; 周鹏; 粟建光
利用随机扩增多态性DNA(RAPD)技术分析了木槿属(Hibiscus)Furcaria组中纤维作物红麻(H.canabinus)及其6个近缘种植物的25份材料.用筛选出的16个引物扩增出192个RAPD条带,它们表现出丰富的多态性.根据得到的RAPD指纹图谱,计算其Nei氏相似系数和遗传距离,并构建了它们的系统树.结果表明:25份材料可划分为7个组,H.penduriformis和H.calyphyllus两个种为一组;红麻种分为两个组,一组为栽培品种,另1组为野生型材料;其余4个种各成为1组.玫瑰麻(H.sabdariffa)和金线吊芙蓉(H.radiatus)的关系较近,而且两者与红麻的亲缘关系也较近,而其它四个种与红麻的关系较远.H.trionum与其它六个种的关系较远.%RAPDs were used to study the relationships among species in Hibiscus sect. Furcaria.Twenty-five accessions of 7 species were examined. Sixteen primers of arbitrary sequences were screened from 80 primers for DNA amplification. A total of 192 bands were generated, of which 149 bands were polymorphic markers. Dendrogram was constructed using Nei's genetic similarity value and MINTS program. The result showed that 25 accessions were clustered in 7 clades, H.penduriformis and H. calyphyllus formed a clade, and 15 accessions of H. cannabinus were clustered in two clades, one being the cultivars, and the other, the wild type materials. The rest 4 species formed 4 individual clades. The most parsimonious dendrogram shows that H. radiatus is closely related to H. sabdariffa. The closely related species to the cultivars of H. cannabinus are H. sabdariffa and H. radiatus, whereas H. trionum appears to be highly diverged from all the other taxa.
The brown planthopper, N. lugens (Staal), has become a serious threat to rice production throughout tropical and sub-tropical Asia with the spread of high yielding rice varieties and intensive culture practices since about 1970. It causes 'hopperburn' and complete wilting and drying of rice plants. Another N. lugens population was found to infest a weed grass, Leersia hexandra that grows abundantly in canals near irrigated rice fields in southeastern Asia. The Leersia infesting N. lugens population fails to survive on rice plants. Conversely, rice infesting N. lugens does not thrive on Leersia. Two sympatric populations of N. lugens, one from rice and other from L. hexandra were collected from five locations in Malaysia. The locations were University Putra Malaysia (UPM), Tanjung Karang (TK), Melaka (MK), Perak (PK) and Sabah (SB). An out group, N. bakeri was also collected from Cameron Highlands (CH). The insects used for long primer RAPD analysis were tested for esterase activity on a simple filter paper using the method reported by Pasteur and Georghiou. DNA extraction and PCR protocols were followed as described by Gillings and Holley. Four Long RAPD primers were used in this study. The primer, pehA no.6, 5'ATCGCACTTGATGCGCAGGCCGTT was diagnostic and the other three yielded the strongest bands and showed polymorphisms in rice and Leersia populations of N. lugens. Cluster analysis based on genetic distance revealed that the 10 brown planthopper populations and an out group, N. bakeri were divided into three major clusters. N. bakeri formed the most isolated cluster from populations of either rice or Leersia infesting populations of N. lugens. The rice infesting populations of five localities clustered together as a group. On the other hand Leersia infesting populations of the same localities formed another distinct cluster. An analysis of molecular variance was also performed and confirmed the differentiation into two groups. One RAPD band that was obtained
Daniel W. H. Robarts
Full Text Available In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR, random-amplified polymorphic DNA (RAPD, and amplified fragment length polymorphism (AFLP to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use. highly variable marker with inherent biological significance.
Hill, Karen K.; Ticknor, Lawrence O.; Okinaka, Richard T.; Asay, Michelle; Blair, Heather; Bliss, Katherine A.; Laker, Mariam; Pardington, Paige E.; Richardson, Amber P.; Tonks, Melinda; Beecher, Douglas J.; Kemp, John D.; Kolstø, Anne-Brit; Wong, Amy C. Lee; Keim, Paul
DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. D...
Kokotovic, Branko; Bolske, G.; Ahrens, Peter; Johansson, K.E.
The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size...... found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes....
Kokotovic, Branko; Friis, N.F.; Ahrens, Peter
Mycoplasma hyosynoviae strains from Denmark, Germany, Japan, Sweden, the Netherlands and the UK were examined for variations in the genomic DNA and within the 16S ribosomal RNA (rRNA) gene. Variations in the chromosomal DNA among 57 isolates recovered from the respiratory tract and joints of pigs...... examined for variation within the 16S rRNA gene. Ten field strains possessed the 16S rDNA sequences identical to the type strain, while the remaining six strains had sequences that differed by one to two nucleotides from that obtained from the type strain....
Low Genetic Diversity Among Garlic (Allium sativum L. Accessions Detected Using Random Amplified Polymorphic DNA (RAPD Escasa Diversidad Genética entre Accesiones de Ajo (Allium sativum L. Detectada Mediante ADN Polimórfico Amplificado al Azar (RAPD
Mario Paredes C
Full Text Available Garlic (Allium sativum L. is a species of vegetative propagation, showing high morphological diversity. Besides, its clones have specific adaptations to different agroclimatic regions. The objective of this study was to determine the genetic diversity of 65 garlic clones collected in Chile and introduced from different countries, by using RAPD (Random Amplified Polymorphic DNA. Fourty random primers of 10 mers generated a total of 398 bands with an 87% of polymorphism. Each primer amplified between two and 20 bands. The size of the fragments obtained fluctuated between 3200 and 369 bp. The results showed that the clones analyzed had a genetic similarity rate of 94%. In addition, 70% of them were clustered in one major group. However, in spite of that situation several clones have different agronomic characteristicsEl ajo (Allium sativum L. es una especie de propagación vegetativa, que presenta una amplia variabilidad morfológica. Los clones de esta especie tienen una adaptación específica a diferentes regiones agroclimáticas. El objetivo de este estudio fue determinar la diversidad genética existente en 65 clones de ajos colectados en Chile e introducidos desde diferentes países, utilizando RAPD (ADN Polimórfico Amplificado al Azar. Para esta evaluación se utilizaron 40 partidores de 10-mers. Los partidores generaron entre dos y 20 bandas, observándose un alto número de patrones con bandas múltiples. Los fragmentos generados difieren en su tamaño entre 3.200 y 369 pb. Los partidores generaron 398 bandas, de las cuales un 87% fueron polimórficas. El análisis estadístico realizado detectó una similitud genética alta, de un 94% entre las accesiones evaluadas, donde aproximadamente un 70% de los clones formaron un grupo homogéneo. Sin embargo, este grupo incluye clones que presentan diferentes características agronómicas
谢震; 冉玉平; 刘瑞; 杨如学; 李志瑜; 代亚玲
目的 用随机扩增多态性DNA方法 研究分离自花斑糠疹的马拉色菌种间和株间差异,了解随机扩增多态性DNA分析(RAPD)与生理生化方法 在菌种分型上的差异及菌株DNA型别和菌种间的关系.方法 用氯化苄法提取马拉色菌标准株(10株7个种)和临床分离株(47株)的基因组DNA,其中临床株分离自34例花斑糠疹患者,经形态学和生理生化方法 鉴定为5个种(合轴马拉色菌、糠秕马拉色菌、钝形马拉色菌、球形马拉色菌、限制马拉色菌),用4种随机引物(S22、SS24、S25、S33)对菌株DNA做PCR随机扩增,NTSYS软件自动生成树状分支图.结果 绝大多数标本均可被4种引物扩增而获得清晰条带,其中2种引物(S22、S24)的条带更为稳定、清晰.共82条DNA片段被扩增,所有菌株均可见种间和株间多态性.有4例患者皮损同时分离出不同种的菌株显示遗传相似性高,在树状图中归入一类.结论 来自同一宿主的不同菌株遗传趋同现象提示马拉色菌的种特异性、菌种演化与宿主间存在密切关系.%Objective To investigate intraspecific and interspecific variation within Malassezia iso-lates from patients with pityriasis versicolor by random amplified polymorphic DNA (RAPD) analysis, to learn the difference between RAPD analysis and physiological and biochemical methods in the typing of Malassezia species, and to explore the relationship between RAPD patterns and Malassezia species. Methods A total of 47 Malassezia isolates were obtained from 34 patients with pityriasis versicolor, and they were classified into 5 species by morphological, physiological and biochemical features, I.e., M. Fin'fur, M. Obtusa, M. Globosa, M. Restricta and M. Sympodialis. Genomic DNA was extracted from the 47 clinical isolates and 10 reference strains (including 7 species) of Malassezia. PCR was performed using 4 random primers including S22, S24, S25 and S33. RAPD patterns were analyzed by NTSYS
Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis
Full Text Available Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST. We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V. braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.
Yu-Tsung Lin; Fuh-Jyh Jan; Chia-Wei Lin; Chien-Hung Chung; Jo-Chu Chen; Shy-Dong Yeh; Hsin-Mei Ku
A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially exp...
侯和胜; 刘洪艳; 佟少明
应用40个引物对中国芦荟(Aloe vera var. chinese),鹿角芦荟库拉索芦荟(Aloe barbadensis L.),木立芦荟皂质芦荟(Aloe saponaria),Aloe vera hybrid 1 ,Aloe vera hybrid 2,皮具刺芦荟朱旺纳芦荟(Aloe juvenna)等9种芦荟的亲缘关系进行了初步分析。其中18个引物的扩增结果具有非常明显的品系间多态性。将结果以Phylip软件包，由程序Neighbor-Joining和UPGMA，分别构建聚类图。结果表明中国芦荟，Aloe vera hybrid 1和皮具刺芦荟具有较近的亲缘。鹿角芦荟，木立芦荟和库拉索芦荟具有较近的亲缘，其中鹿角芦荟，木立芦荟的遗传距离特别近，可认为是同一品种的变种，由于形态上的一些分化而出现了不同的命名。皂质芦荟，Aloe vera hybrid2 的亲缘关系较近。朱旺纳芦荟与各品种之间都保持着相当远的遗传距离，原因可能是该品种未广泛栽培，从而还保持着某些自己的遗传特性。%The phylogenetic relationship of nine Aloes,including Aloe vera var. chinese, Aloe arborescens var. natalensis bgr. ,Aloe barbadensis L. ,Aloe arboreseens , Aloe saponaria , Aloe vera hybrid 1, Aloe vera hybrid 2, Aloe aculeata and Aloe juvenna was investigated by RAPD analysis. 18 single primers were selected out of 40 random primers. The amplified fragments of 18 primers were analyzed by the Phylip software packet, and the phylogenetic trees were constructed by UPGMA method and N-J method respectively. The results indicated as the following: Aloe vera var. chinese, Aloe vera hybrid 1 and Aloe aculeata have close phylogenetic relationship. Aloe arborescens var. natalensis bgr., Aloe barbadensis L. and Aloe arborescens have close phylogenetic relationship. The phylogenetic relationship between Aloe arborescens and Aloe arborescens was much closer. Aloe saponaria and Aloe vera hybrid 2 have close phylogenetic relationship too. The phylogenetic relationship of Aloe juvenna was far away from the others.
Kokotovic, Branko; Friis, N.F.; Jensen, J.S.;
Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including......I restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp, The method was able to...
Dendrobium Sonia white mutant lines were obtained through gamma ray induced mutation of purple flower Dendrobium Sonia at dosage 35 Gy. Amplified Fragment Length Polymorphism (AFLP) technique was used to compare genomic variations in these mutant lines with the control. Our objectives were to detect polymorphic fragments from these mutants to provide useful information on genes involving in flower colour expression. AFLP is a PCR based DNA fingerprinting technique. It involves digestion of DNA with restriction enzymes, ligation of adapter and selective amplification using primer with one (pre-amplification) and three (selective amplification) arbitrary nucleotides. A total number of 20 primer combinations have been tested and 7 produced clear fingerprint patterns. Of these, 13 polymorphic bands have been successfully isolate and cloned. (Author)
Huiquan Zheng; Hongjing Duan; Dehuo Hu; Ruping Wei; Yun Li
Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is one of the most important coniferous tree species used for timber production in China. Here, we conducted a sequence-related amplified polymorphism (SRAP) primer screening assay with a total of 594 primer combinations, using 22 forward and 27 reverse primers on four repre-sentative Chinese fir genotypes. The obtained results indicated that Chinese fir genomic DNA has a notable amplification bias on the employed forward or reverse primer nucleotides (3' selection bases). Out of the tested primer sets, 35 primer combinations with clearly distin-guished bands, stable amplification, and rich polymorphism were selected and identified as optimal primer sets. These optimal primer pairs gave a total of 379 scorable bands, including 265 polymorphic bands, with an average of 10.8 bands and 7.6 polymorphic bands per primer combination. The produced band number for each optimal primer set ranged from 7 to 14 with a percentage of polymorphic bands spanning from 33.3 to 100.0%. These primer combinations could facilitate the next SRAP analysis assays in Chinese fir.
Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus Otimização do protocolo de amplificação randômica de DNA polimórfico para identificação molecular de Lophius gastrophysus
Micheline S. Ramella
Full Text Available Lophius gastrophysus has important commercial value in Brazil particularly for foreign trade. In this study, we described the optimization of Random Amplified Polymorphic DNA (RAPD protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl concentrations, DNA quantity were tested to find reproducible and adequate profiles. Amplifications performed with primers A01, ² A02 and A03 generate the best RAPD profiles when the conditions were annealing temperature of 36ºC, 25 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary and RAPD could be used as an accurate, rapid tool to expose commercial fraud.Lophius gastrophysus apresenta importante valor comercial no Brasil, principalmente para a exportação. Neste estudo, descrevemos uma otimização do protocolo de amplificação aleatória de DNA polimórfico (RAPD para identificação de L. gastrophysus. Diferentes condições (temperatura de recozimento, quantidade de DNA e concentração de MgCl2 foram testadas para obter perfis reprodutíveis. Os iniciadores A01, A02 e A03 geraram os melhores resultados de amplificação quando utilizados temperatura de recozimento de 36ºC, 25 ng de DNA e 2,5 mM de MgCl2. A identificação exata de espécies e da origem dos produtos marinhos faz-se necessária e a RAPD é uma ferramenta rápida e precisa para expor fraudes comerciais.
野蘑菇(Agaricus arvensis Schaeff ex.Fr.)是蘑菇属(Agaricus)又一种近年来广泛栽培的食用菌.由于它的菌丝细胞具有多核,无锁状联合,以及人们对它的繁殖模式和生活史认识的不足,给杂交育种工作造成了较多困难.运用随机扩增多态DNA遗传标记技术,结合拮抗试验和核相分析,对自育的单孢菌株之间的杂交试验进行分析研究.结果表明:两个相互亲合的同核体菌株被配对培养时,交配反应出现,并形成异核体的后代.可能野蘑菇具有双重的交配繁殖系统--同宗配合和异宗配合.在食用菌杂交育种研究中,机扩增多态DNA(RAPD)是一种非常有效和方便的检验杂合子的方法.
Osentoski, M F; Lamb, T
The slow rate of mtDNA evolution in turtles poses a limitation on the levels of intraspecific variation detectable by conventional restriction fragment surveys. We examined mtDNA variation in the gopher tortoise (Gopherus polyphemus) using an alternative restriction assay, one in which PCR-amplified segments of the mitochondrial genome were digested with tetranucleotide-site endonucleases. Restriction fragment polymorphisms representing four amplified regions were analysed to evaluate population genetic structure among 112 tortoises throughout the species' range. Thirty-six haplotypes were identified, and three major geographical assemblages (Eastern, Western, and Mid-Florida) were resolved by UPGMA and parsimony analyses. Eastern and Western assemblages abut near the Apalachicola drainage, whereas the Mid-Florida assemblage appears restricted to the Brooksville Ridge. The Eastern/Western assemblage boundary is remarkably congruent with phylogeographic profiles for eight additional species from the south-eastern U.S., representing both freshwater and terrestrial realms. PMID:8564009
Sasha A Langley
Full Text Available An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures . Nucleosome sequences isolated from diverse organisms exhibit ∼10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these
van der Zee, Anneke; Steer, Niels; Thijssen, Eveline; Nelson, Jolande; van't Veen, Annemarie; Buiting, Anton
We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal resul...
Sugar cane is the fourth most important cash crop of Pakistan and is grown on 1 million hectares of land, with a total production of 37 million tonnes. It does not flower under existing environmental conditions. Sugar cane is vegetatively propagated and the national breeding programmes is restricted to the adaptation and multiplication of exotic varieties. Random amplified polymorphic DNA (RAPD) markers were used to establish polymorphisms among various local sugar cane varieties. DNA from the varieties L-118, L-116, BL-4, BF-162, Col-44, Col-54, Triton and Puri was isolated and amplified by polymerase chain reaction using ten nucleotide primers. The amplification profiles of all the sugar cane varieties were compared and the polymorphisms detected. DNA was isolated from the embryogenic calli of sugar cane subjected to gamma irradiation at different doses (0, 0.5, 2.0, 4.0 and 6.0 krad) and salt stresses (NaCl: 0, 50, 100, 150 and 200 mM), and was amplified with random primers to detect the polymorphisms introduced by stress. The banana is another important vegetatively propagated crop in Pakistan. DNA isolation from micropropagated banana was optimized and RAPD analysis performed on several clones of the banana variety Williams. The level of genetic variability revealed from calli and vegetatively propagated sugar cane and banana by RAPD analysis is discussed. (author). 10 refs, 5 figs, 2 tabs
WU Jian; SUN Ri-fei; ZHANG Yan-guo; WANG Xiao-wu
Ecotilling is a new approach based on enzyme-mediated heteroduplex cleavage to discover DNA polymorphisms in natural population. We used mung bean nuclease(MBN) instead of routinely used CELI to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we added M13 adapter to 5'end of gene specific primers to make IRD dye labeled M13 forward and reverse primers possibly universal for different genes. A Brassica rapa ZIP gene homologue was subjected to the analysis to practise the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.
Khush, R S; Becker, E; Wach, M
Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus. PMID:1444410
Full Text Available Aquilaria sp. (Thymelaeaceae is the most valuable non wood production of forestry plant in Indonesia. It produces a fragrant resin when subjected to fungal attack and has been traded internationally known as gaharu. Knowledge of genetic diversity and relationship among species and genus is important for breeding purposes and species conservation. In this study, genetic variabilityof six Aquilaria species were analyzed using the AmplifiedFragment Length Polymorphism (AFLP markers. Ten AFLP primercombinations amplified 1353 DNA fragments ranging in size from100 to 350 bp of which 1285 (95% of them were polymorphic. Genetic similarities among Aquilaria sp. consisted of A. malaccensis, A. beccariana, A. microcarpa, and A. crassna ranged from 63.90 to 72.00 % based on Dice coefficient. The dendrogram derivedby the unweighted pair group method with arithmetic mean of germplasm analysis were clustered into two main groups. Hence, a genetic variation among species is quiet high. Bootstrap valuesfor the groups supported 70% of the cluster using a linear relationship equation of (r = 0.724, P < 0.0001 was observedbetween known pedigrees and AFLP-derived genetic similarityfor 136 pairwise comparisons of Aquilaria species. For example, A. malacensis and A. microcarpa have the highest genetic similarity (72.00% compared with another Aquilaria species. Primer pairs E-ACG/M-CTA produced a specific fragment for A. beccariana (850 bp, A. crasna (550 bp, 180 bp, and 140 bp, A. malaccencis (1500 bp, A. microcarpa (250 bp and Gyrinops versteegii (150 bp. Primer pairs E-ACG/M-CAA produced a specific DNA fragment only for A. beccariana (1500 bp and 100 bp. Primer pairs E-ACC/M-CAC also produced only specific fragment for A. crassna (1500 bp. Study showed the usefulness of AFLPanalysis in Aquilaria sp. and its potential application for breedingand species conservation. Further, molecular diversity estimated in the present study combined with the datasets on other
目的 采用随机扩增多态性DNA(RAPD)基因分型方法 监测肺炎克雷伯菌医院感染情况. 方法 对我院神经外科重症监护室临床分离的72株肺炎克雷伯菌进行RAPD基因分型,并通过指纹图谱比较分析,确证医院感染爆发. 结果 72株肺炎克雷伯菌共分得68型,未发生肺炎克雷伯菌医院感染局部流行. 结论 RAID基因分型方法 可对肺炎克雷伯菌进行分子流行病学调查.%Objective To monitor nosocomial infections of Klebsiella pneumoniae by analyzing the randomly amplified polymorphic DNA (RAPD). Methods RAPD analysis was used to genotype 72 Klebsiella pneumoniae strains isolated from the intensive care unit (ICU) of the neurosurgical department, and the fingerprints were compared to confirm the outbreak of nosocomial infection of Klebsiella pneumoniae. Results 72 strains Klebsiella pneurnoniae were classified into 68 RAPD types, suggesting the absence of an outbreak of nosocomial infection of glebsiella pneurnoniae in the ICU.Conclusian RAPD genotyping can be used in the molecular epidemiological study ofKlebsiella pneurnoniae.
Møller, Kristian; Jensen, Tim Kåre; Boye, Mette; Leser, T.D.; Ahrens, Peter
Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due to the...
On, Stephen L.W.; Atabay, H.I.; Amisu, K.O.;
Aims: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. Methods and Results: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and Nor...
Hong, Y; Garcia, M.; Levisohn, S; Lysnyansky, I.; Leiting, V.; Savelkoul, P. H. M.; Kleven, S. H.
Amplified fragment length polymorphism (AFLP) was used for typing avian mycoplasma species. Forty-four avian mycoplasma strains were successfully typed into eight distinct groups, with each representing a different species. Homology of AFLP patterns of 35% or less was used as a cutoff value to differentiate avian mycoplasma strains into different species.
Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.
Studer, Bruno; Jensen, Louise Bach; Fiil, Alice;
High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting...... curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved...... in vernalization response successfully discriminated genotypes in absence of allelic sequence information, and allowed to determine allele segregation in VrnA. Here we introduce the concept of "blind" mapping based on HRM as a powerful, fast and cheap method to map any DNA sequence polymorphisms without prior...
Xiong, Jie; Tao, Tao; Zheng, Xiaoguo; Wei, Haibin; Yue, Yunxia; Chen, Liang; Luo, Lijun
The stress-induced epimutations could be inherited over generations and play important roles in plant adaption to stressful environments. Upland rice has been domesticated in water-limited environments for thousands of years and accumulated drought-induced epimutations of DNA methylation, making it epigenetically differentiated from lowland rice. To study the epigenetic differentiation between upland and lowland rice ecotypes on their drought-resistances, the epigenetic variation was investigated in 180 rice landraces under both normal and osmotic conditions via methylation-sensitive amplified polymorphism (MSAP) technique. Great alterations (52.9~54.3% of total individual-locus combinations) of DNA methylation are recorded when rice encountering the osmotic stress. Although the general level of epigenetic differentiation was very low, considerable level of ΦST (0.134~0.187) was detected on the highly divergent epiloci (HDE). The HDE detected in normal condition tended to stay at low levels in upland rice, particularly the ones de-methylated in responses to osmotic stress. Three out of four selected HDE genes differentially expressed between upland and lowland rice under normal or stressed conditions. Moreover, once a gene at HDE was up-/down-regulated in responses to the osmotic stress, its expression under the normal condition was higher/lower in upland rice. This result suggested expressions of genes at the HDE in upland rice might be more adaptive to the osmotic stress. The epigenetic divergence and its influence on the gene expression should contribute to the higher drought-resistance in upland rice as it is domesticated in the water-limited environment. PMID:27380174
Fulneček, Jaroslav; Kovařík, Aleš
Roč. 15, JAN 2014 (2014). ISSN 1471-2156 R&D Projects: GA ČR(CZ) GBP501/12/G090; GA ČR(CZ) GA13-10057S; GA ČR(CZ) GA206/09/1751 Institutional support: RVO:68081707 Keywords : MSAP * DNA methylation * Methylcytosine Subject RIV: BO - Biophysics Impact factor: 2.397, year: 2014
Ejaz, M; Qidi, Z; Gaisheng, Z; Na, N; Huiyan, Z; Qunzhu, W
Amplified fragment length polymorphism markers were used to assess genetic diversity in 10 male sterile wheat crop lines (hetero-cytoplasm with the same nucleus) in relation to a restorer wheat line. These male sterile lines were evaluated using 64 amplified fragment length polymorphism primer combinations, and 13 primers produced polymorphic bands, generating a total 682 fragments. Of the 682 fragments, 113 were polymorphic. The polymorphic information content and marker index values demonstrated the utility of the primer combinations used in the present study. Unweighted pair group method with arithmetic mean and principal coordinate analysis of the genotypic data revealed clustering of accessions based on genetic relationships, and accessions were separated into 2 groups with their restorer line. Jaccard's similarity coefficient values suggested good variability among the male sterile lines, indicating their utility in breeding programs. The fallouts of analysis of molecular variance showed large within-group population variation, accounting for 77% of variation, while among-group comparison accounted for 23% of the total molecular variation, which was statistically significant. The molecular diversity observed in this study will be useful for selecting appropriate accessions for plant improvement and hybridization through molecular-breeding approaches and for developing suitable conservation strategies. PMID:26345825
Li, Cheng-Tao; Li, Li
Amplified fragment length polymorphism (AFLP) is a new molecular marker to detect genomic polymorphism. This new technology has advantages of high resolution, good stability, and reproducibility. Great achievements have been derived in recent years in AFLP related technologies with several AFLP expanded methodologies available. AFLP technology has been widely used in the fields of plant, animal, and microbes. It has become one of the hotspots in Forensic Botany. This review focuses on the recent advances of AFLP and its applications in forensic biology. PMID:18979924
Objective To investigate the mitochondrial DNA sequence polymorphism sites in Chinese YUGU ethnic group and to provide basic data used in forensic purpose. Methods Genomic DNA was extracted from the hole blood of 100 unrelated individuals of Chinese YUGU ethnic group by standard chelex-100 method. The sequence polymorphism sites was determined by PCR amplification and direct sequencing. Results 54 polymorphic sites were noted in mtDNA np16091-16418 region, and 46 haplotypes were identified. The genetic diversity was calculated to be 0.9691, and the genetic identity was calculated to be 0.0406. Conclusion There are some particular polymorphism sites in Chinese YUGU ethnic group. The results suggest that sequence polymorphism from np16091-16418 in human mitochondrial DNA can be used as a biological marker for forensic identity.
Rai, Ved Prakash; Kumar, Rajesh; Kumar, Sanjay; Rai, Ashutosh; Kumar, Sanjeet; Singh, Major; Singh, Sheo Pratap; Rai, Awadesh Bahadur; Paliwal, Rajneesh
A sound knowledge of the genetic diversity among germplasm is vital for strategic germplasm collection, maintenance, conservation and utilisation. Genomic simple sequence repeats (SSRs) and random amplified microsatellite polymorphism (RAMPO) markers were used to analyse diversity and relationships among 48 pepper (Capsicum spp.) genotypes originating from nine countries. These genotypes covered 4 species including 13 germplasm accessions, 30 improved lines of 4 domesticated species and 5 lan...
NURITA TORUAN-MATHIUS; DEWI RAHMAWATI; ANIDAH
Aquilaria sp. (Thymelaeaceae) is the most valuable non wood production of forestry plant in Indonesia. It produces a fragrant resin when subjected to fungal attack and has been traded internationally known as gaharu. Knowledge of genetic diversity and relationship among species and genus is important for breeding purposes and species conservation. In this study, genetic variabilityof six Aquilaria species were analyzed using the AmplifiedFragment Length Polymorphism (AFLP) markers. Ten ...
Kassama, Yankuba; Rooney, Paul J.; Goodacre, Royston
The ability of the fluorescent amplified fragment length polymorphism (FAFLP) technique to identify bacterial isolates from urinary tract infections (UTIs) was investigated. FAFLP was carried out using the single primer combination MseI plus CT and EcoRI plus 0, and information-rich FAFLP profiles were generated from all 69 UTI isolates studied, which comprised both gram-negative and gram-positive bacteria encompassing eight genera. The genetic relatedness of these 69 bacteria was determined ...
Lawson, Andrew J.; Stanley, John; Threlfall, E. John; Desai, Meeta
Fluorescent amplified fragment length polymorphism (FAFLP) subtyping analysis was used to genotype multiresistant Salmonella enterica serovar Typhimurium definitive phage type 104. Thirteen distinct FAFLP profiles were found among 85 isolates exhibiting identical pulsed-field gel electrophoresis (PFGE) profiles. A single FAFLP profile was shared by 93% of outbreak-associated isolates and 82% of sporadic isolates. This study demonstrates the value of FAFLP as a high-resolution tool for epidemi...
Bendhifi Zarroug, M; Baraket, G; Zourgui, L; Souid, S; Salhi Hannachi, A
Opuntia ficus indica is one of the most economically important species in the Cactaceae family. Increased interest in this crop stems from its potential contribution to agricultural diversification, application in the exploitation of marginal lands, and utility as additional income sources for farmers. In Tunisia, O. ficus indica has been affected by drastic genetic erosion resulting from biotic and abiotic stresses. Thus, it is imperative to identify and preserve this germplasm. In this study, we focused on the use of random amplified microsatellite polymorphisms to assess genetic diversity among 25 representatives of Tunisian Opuntia species maintained in the collection of the National Institute of Agronomic Research of Tunisia. Seventy-two DNA markers were screened to discriminate accessions using 16 successful primer combinations. The high percentage of polymorphic band (100%), the resolving power value (5.68), the polymorphic information content (0.94), and the marker index (7.2) demonstrated the efficiency of the primers tested. Therefore, appropriate cluster analysis used in this study illustrated a divergence among the cultivars studied and exhibited continuous variation that occurred independently of geographic origin. O. ficus indica accessions did not cluster separately from the other cactus pear species, indicating that their current taxonomical classifications are not well aligned with their genetic variability or locality of origin. PMID:25730081
Fearnley, C.; On, S.L.W.; Kokotovic, Branko; Manning, G.; Cheasty, T.; Newell, D. G.
An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y enterocolitica strains according to th...
许顺斌; 黄尚志; 罗会元
Four (CA), repeats, located in introns,44,45,49 and 50 of the dystrophin gene,were evaluated in Chinese.These loci are highly polymorphic,with polymorphism information contents of 0.872,0.772,0.870 and 0.718,respectively.All four loci can be easily amplified and labelled using two duplex PCR reactions with α-32P-dCTP and can be detected by denaturing polyacrylamide gel electrophoresis.Using these four loci and the two polymorphic(CA)n repeats located at the 5′ and 3′ ends of the dystrophin gene,we have developed a new PCR-based procedure-Amp-FLP( amplified fragment length polymorphism)linkage analysis for the gene diagnosis of DMD/BMD.This method can detect intragenic recombination rapidly and efficiently and greatly improves the success rate of carrier deterction and prenatal diagnosis in non-deletion DMD/BMD families.All of the loci used in this procedure are intragenic.In addition ,the loci in introns 44,45,49 and 50 are located in the deletion-prone region of the dystrophin gene,making them valuable and usefui in the identification of deletion mutations.Here we report one case of deletion detection using these four loci.
Torres, Rogelio R; Arias, Maria C; Moretto, Geraldo
The geographical distribution of the Brazilian endemic stingless bee Melipona quadrifasciata quadrifasciata Lepeletier ranges from Rio Grande do Sul to Minas Gerais states. The objective of the present study was to verify mtDNA polymorphisms among samples of M. q. quadrifasciata collected in southern Brazil. Twenty nine colonies from three localities (Blumenau and Mafra/SC and Prudentópolis/ PR) were sampled. Seven mtDNA regions were amplified and further digested with 15 restriction enzymes (PCR-RFLP). Five composite haplotypes were identified, with two unique to samples from Prudentópolis and the remaining three to samples from Mafra and/or Blumenau. PMID:19488509
王苏建; 王家平; 王仕忠; 鲁科峰; 钱丽萍
Fabiola MORENO-OLIVAS; Vincent U.GANT Jr; Kyle L.JOHNSON; Jose R.PERALTA-VIDEA; Jorge L.GARDEA-TORRESDEY
重要结论：采用随机扩增多态性DNA技术，发现TiO2纳米颗粒污染处理的西葫芦样品与未处理样品的基因组DNA图谱相比，不仅在谱带强度有明显差异，而且存在谱带消失和新谱带产生现象，表明TiO2纳米颗粒对西葫芦具有基因毒性。%Titanium dioxide nanoparticles (TiO2 NPs) are used in cosmetics, sunscreens, paints, and toothpaste, among other applications. These NPs are very stable and can be transported and dispersed in wastewater and biosolids. Animal species have shown negative reactions to TiO2 NPs. However, little is known about their toxicity in plants, specifically the possibility of gen-otoxic effects. In this study, we used a random amplified polymorphic DNA (RAPD) technique to study the genotoxic effects of TiO2 NPs on hydroponically cultivated zucchini (Cucurbita pepo) plants. Seeds were allowed to germinate for 7 d and plants were selected at random for individual and population studies. Four plants were selected for the individual study and 18 for the popu-lation study. RAPD profiles of TiO2 NPs treated plants showed differences in band intensity, loss of bands, or appearance of new bands, compared to untreated plants. To the authors’ knowledge, this is the first report of the genotoxic potential of TiO2 NPs in zucchini.
Mochizuki, Takashi; Ishizaki, Hiroshi; Barton, Richard C.; Moore, Mary K.; Jackson, Colin J.; Kelly, Steven L.; Evans, E. Glyn V.
Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile...
Prince, J P; Lackney, V K; Angeles, C; Blauth, J R; Kyle, M M
Interspecific genetic variation was examined in the genus Capsicum based on shared restriction fragments in Southern analyses. Four distinct clusters were delineated among 21 accessions of cultivated and wild pepper (C. annuum, C. baccatum, C. chacoense, C. chinense, and C. frutescens). Three tight clusters comprised of accessions belonging to C. annuum, C. frutescens, and C. baccatum, respectively, were formed, along with a fourth cluster comprised of one accession each of C. chinense and C. chacoense. All accessions were differentiated by this technique, and the clusters corresponded closely to previous morphology-based classification. Sufficient DNA polymorphism exists among these accessions that segregating populations useful for restriction fragment length polymorphism (RFLP) mapping could be constructed using any two pepper accessions as parents. Regression analysis indicates that genetic distance is a good predictor (R2 = 0.872) of the level of mappable DNA polymorphism in Capsicum. Intraspecific variability was examined among four C. annuum cultivars (NuMex R Naky, Jupiter, Perennial, and Criollo de Morelos 334) using both RFLPs and randomly amplified polymorphic DNA (RAPDs), allowing a comparative evaluation of the two techniques. Seventeen percent of the clones used singly in RFLP analyses were sufficient for the differentiation of these varieties, as were 12.5% of the RAPD PCR amplifications. Dendrograms constructed from RFLP and RAPD analyses of the intraspecific data are similar but not identical. Southern analysis and RAPD PCR should be useful for DNA fingerprinting and the discrimination of closely related C. annuum genotypes. PMID:7774796
叶明亮; 李晓娣; 公衍文; 侯晓琦; 吕波; 薛燕; 袁著忻
Full Text Available Identification and characterization of genotypes is essential for improving the quality of cultivated varieties in breeding programs. Information about the origin of varieties can help farmers in selecting appropriate varieties to specific growing conditions or end use of crops. A set of ten microsatellite markers was used to describe genetic diversity in a sample of 30 barley (Hordeum vulgare L. genotypes. A total of 55 different alleles were amplified using ten SSR markers localized on chromosomes 1H, 2H, 3H, 5H, 6H, 7H with an average number of 5.5 alleles per locus. On the basis of allele frequencies we have calculated diversity index, polymorphic information content and index of probability, which have mean values of 0.664; 0.643 and 0.126 respectively. These values indicate high differentiation ability of SSR markers. In the created dendrogram using hierarchical cluster analysis using UPGMA algorithm we were able to differentiate all 30 barley genotypes. The results show that DNA markers are suitable for the identification and differentiation of genotypes and indicated the effectiveness of microsatellite markers to describe genetic diversity.
Khaled Chatti; Olfa Saddoud; Amel Salhi-Hannachi; Messaoud Mars; Mohamed Marrakchi; Mokhtar Trifi
The random amplified mirosatellite polymorphism method was performed in a set of Tunisian fig landraces using eighteen primer combinations. Atotal of sixty three random amplified microsatellite polymorphism (RAMPO) markers were scored and used either to assess the genetic diversity in these cultivars or to detect cases of mislabeling. Opportunely, data proved that the designed procedure constitutes an attractive and fast method with low costs and prevents radio exposure. As a result, we have identified the primer combinations that are the most efficient to detect genetic polymorphism in this crop. Therefore, the derived unweighted pair-group method with arithmetic averages (UPGMA) dendrogram illustrates the genetic divergence among the landraces studied and exhibits a typically continuous variation. Moreover, no evident correlation between the sexes of trees was observed. In addition, using these markers, discrimination between landraces has been achieved. Thus, random amplified mirosatellite polymorphism is proved to be powerful for characterizing the local fig germplasm.
Rai, Ved Prakash; Kumar, Rajesh; Kumar, Sanjay; Rai, Ashutosh; Kumar, Sanjeet; Singh, Major; Singh, Sheo Pratap; Rai, Awadesh Bahadur; Paliwal, Rajneesh
A sound knowledge of the genetic diversity among germplasm is vital for strategic germplasm collection, maintenance, conservation and utilisation. Genomic simple sequence repeats (SSRs) and random amplified microsatellite polymorphism (RAMPO) markers were used to analyse diversity and relationships among 48 pepper (Capsicum spp.) genotypes originating from nine countries. These genotypes covered 4 species including 13 germplasm accessions, 30 improved lines of 4 domesticated species and 5 landraces derived from natural interspecific crosses. Out of 106 SSR markers, 25 polymorphic SSR markers (24 %) detected a total of 76 alleles (average, 3.04; range, 2-5). The average polymorphic information content (PIC) was 0.69 (range, 0.29-0.92). Seventeen RAMPO markers produced 87 polymorphic fragments with average PIC of 0.63 (range, 0.44-0.81). Dendrograms based on SSRs and RAMPOs generated two clusters. All 38 Capsicum annuum genotypes and an interspecific landrace clustered together, whereas nine non-annuum (three Capsicum frutescens, one Capsicum chinense, one Capsicum baccatum and four interspecific landraces) genotypes clustered separately. Genetic variation within non-annuum genotypes was greater than the C. annuum genotypes. Distinctness of interspecific derivative landraces grown in northeast India was validated; natural crossing between sympatric Capsicum species has been proposed as the mechanism of their origin. PMID:24431527
Characteristics of polymorphic sequences of DNA, especially satellite, mini satellite and micro satellite sequences are presented. Own experience from the use of multi and single locus analysis of DNA in paternity testing has been compared with the results of research in other laboratories. Critical points of both types of analysis are discussed. (author). 53 refs, 4 figs, 2 tabs
Eel family is a huge one, in which many kinds of eels especially some migratory eels, bear strong resemblance to each other, and are therefore difficult to be identified. In this study 29 random primers were used to make RAPD analysis for Japaneses eel (Anguilla japonica), European eel (Anguilla anguilla) and Pike eel (Muraenesox cinereus).And totally 299 fragments were counted.Shared or specific fragments were counted and genetic similarity or genetic distance were calculated.The genetic similarity between Japanese eel and Pike eel is 0.68 and the genetic distance between them is 0.32;those between European eel and Pike eel are 0.72 and 0.28 respectively,and between Japanese eel and European eel are 0.74 and 0.25 respectively.The method has been shown to be suitable to molecular identification of eels.It provides an alternative approach to determine the relationship between species.
Robert R McWilliams; William R Bamlet; Cunningham, Julie M.; Goode, Ellen L.; de ANDRADE, MARIZA; Lisa A Boardman; Petersen, Gloria M.
Base excision repair and nucleotide excision repair are vital responses to multiple types of DNA damage, including damage from tobacco exposure. Single-nucleotide polymorphisms (SNP) in these pathways may affect DNA repair capacity and therefore influence risk for cancer development. We performed a clinic-based, case-control study comprising 481 consecutive patients with confirmed pancreatic adenocarcinoma and 625 healthy controls. Allele and genotype frequencies for 16 SNPs in DNA repair gen...
Dobner, P; Löscher, Thomas; Rinder, H
A pair of degenerate polymerase chain reaction (PCR) primers (LEI-1, TCG GAT CC[C,T] [G,C]TG GGT AGG GGC GT; LEI-2, ACG GAT CC[G,C] [G,C][A,C]C TAT [A,T]TT ACA CC) defining a 0.15-kb segment ofLeishmania minicircle DNA was constructed. These primers amplified not only inter- but also intraspecifically polymorphic sequences. Individual sequences revealed a higher intraspecific than interspecific divergence. It is concluded that individual sequences are of limited relevance for species determin...
Several DNA fingerprinting techniques that use arbitrary primers to characterize, scan and tag genomic DNA were optimized and used to study plants and microbial pathogens. The generated arbitrarily amplified DNA (AAD) profiles could be tailored in their complexity and polymorphic content, allowing analysis of closely related organisms, such as vegetatively-propagated horticultural crops or clonal fungal populations. AAD markers were used in cultivar and strain identification, map-based cloning, and marker-assisted breeding, sometimes as sequence-tagged sites. Phenetic analysis using parsimony, cluster, and numerical methods was applied successfully to the identification of genetic relationships in turfgrass species such as bermudagrass, woody plants such as dogwoods, and floricultural species such as petunia and chrysanthemum. AAD profiles were used to measure for the first time a genome-wide mutation rate, directly in a plant. Mutation rates in vegetatively propagated bermudagrass were comparable to those in human, mice, fruit flies, and worms. In combination with established tools used in molecular systematics (e.g. rDNA sequence analysis), AAD markers tracked the introduction of exotic dogwood anthracnose-causing fungi in North America. As part of a breeding effort to combat dogwood diseases, AAD was used in pseudo-testcross mapping of the tree at the intra-specific level. Markers were efficiently generated despite the close relatedness of parental dogwood material. Finally, DNA markers and tags were also generated in soybean, and were used to construct high density maps and walk towards defined genomic regions in the positional cloning of the supernodulation nts-1 symbiotic gene. (author)
Full Text Available Leptin is a protein involved in the regulation of feed intake, fat metabolism, whole body energy balance, reproduction and hematopoiesis. In cattle Leptin gene has been considered a potential QTL influencing several production traits like meat production, milk performance and reproduction. Several studies on bovine leptin gene have found association between polymorphisms and traits like milk yield, feed intake, fat content, carcass and meat quality. With the aim to assess the presence of sequences polymorphisms in the Buffalo leptin gene, we sequenced the entire coding region and part of the introns on a panel of Italian River Buffalos. In this study we identified a new set of SNP (Single Nucleotide Polymorphism useful for association studies.
Markina, N V; Usatov, A V; Logacheva, M D; Azarin, K V; Gorbachenko, C F; Kornienko, I V; Gavrilova, V A; Tihobaeva, V E
The polymorphism of microsatellite loci of chloroplast genome in six Helianthus species and 46 lines of cultivated sunflower H. annuus (17 CMS lines and 29 Rf-lines) were studied. The differences between species are confined to four SSR loci. Within cultivated forms of the sunflower H. annuus, the polymorphism is absent. A comparative analysis was performed on sequences of the cpDNA inbred line 3629, line 398941 of the wild sunflower, and the American line HA383 H. annuus. As a result, 52 polymorphic loci represented by 27 SSR and 25 SNP were found; they can be used for genotyping of H. annuus samples, including cultural varieties: twelve polymorphic positions, of which eight are SSR and four are SNP. PMID:26601486
Accurate identifying of the genotypes is considered one of the most important mechanisms used in the recording or the protection of plant varieties. The investigation was conducted at the experimental form belonging to the egyptian Atomic Energy Authority, Inshas. The aim was to evaluate grain quality characteristics and molecular genetic variation using Amplified Fragment Length Polymorphism (AFLP) technique among six rice genotypes, Egyptian Jasmine aromatic rice cultivar and five aromatic rice mutants in (M3 mutagenic generation). Two mutation (Egy22 and Egy24) were selected from irradiated Sakha 102 population with 200 and 400Gy of gamma rays in the M2 generation, respectively, and three mutations ( Egy32, Egy33, and Egy34) were selected from irradiated Sakha 103 population with 200, 300, 400Gy of gamma rays in the M2 generation, respectively. The obtained results showed that the strong aroma was obtained for mutant Egy22 as compared with Egyptian Jasmine rice cultivar (moderate aroma). Seven primer combinations were used through six rice genotypes on the molecular level using AFLP marker. The size of AFLP Fragments Were Ranged from 51- 494bp. The total number of amplified bands was 997 band among them 919 polymorphic bans representing 92.2%. The highest similarity index (89%) was observed between Egyptian Jasmine and Egy32 followed by (82%) observed between Egyptian Jasmine and Egy34. On the other hand, the lowest similarity index was (48%) between Egyptian Jasmine and Egy24. In six rice genotypes, Egy24 produced the highest number of the AFLP makers giving 49 unique markers (23 positive and 26 negative), then Egy22 showed 23 unique markers (27 positive and 6 negative) while Egy33 was characterized by 17 unique markers (12 positive and 5 negative). At last Egyptian Jasmine was discriminated by the lowest number of markets, 10 (6 positive and 4 negative). The study further confirmed that AFLP technique was able to differentiate rice genotypes by a higher number
Zhao, Cui; Liu, Cui; Li, Wei; Chi, Shan; Feng, Rongfang; Liu, Tao
Restriction site amplified polymorphism (RSAP) was used, for the first time, to analyze the genetic structure and diversity of four, mainly cultivated, varieties of the brown alga, Saccharina japonica. Eighty-eight samples from varieties " Rongfu ", " Fujian ", " Ailunwan " and " Shengchanzhong " were used for the genetic analyses. One hundred and ninety-eight bands were obtained using eight combinations of primers. One hundred and ninety-one (96.46%) were polymorphic bands. Nei's genetic diversity was 0.360, and the coefficient of genetic differentiation was 0.357. No inbreeding-type recession was found in the four brown alga varieties and the results of the " Ailunwan " variety using samples from 2 years showed that the variety was becoming less diverse during the selection inherent in the breeding program. Genetic diversity and cluster analyses results were consistent with these genetic relationships. The results show the RSAP method is suitable for genetic analysis. Continuous inbreeding and selection could reduce the genetic diversity effectively; therefore periodical supervision is required.
ZHAO Cui; LIU Cui; LI Wei; CHI Shan; FENG Rongfang; LIU Tao
Restriction site amplified polymorphism (RSAP) was used,for the first time,to analyze the genetic structure and diversity of four,mainly cultivated,varieties of the brown alga,Saccharinajaponica.Eighty-eight samples from varieties "Rongfu","Fujian","Ailunwan" and "Shengchanzhong" were used for the genetic analyses.One hundred and ninety-eight bands were obtained using eight combinations of primers.One hundred and ninety-one (96.46％) were polymorphic bands.Nei's genetic diversity was 0.360,and the coefficient of genetic differentiation was 0.357.No inbreeding-type recession was found in the four brown alga varieties and the results of the "Ailunwan" variety using samples from 2 years showed that the variety was becoming less diverse during the selection inherent in the breeding program.Genetic diversity and cluster analyses results were consistent with these genetic relationships.The results show the RSAP method is suitable for genetic analysis.Continuous inbreeding and selection could reduce the genetic diversity effectively; therefore periodical supervision is required.
Full Text Available Problem statement: Rhizoctonia solani is a potential grapevine pathogen. In order to develop effective methods of control, it is necessary to document its genetic diversity. Approach: The objective of this study was to analyze the genetic variability of R. solani isolated from the rhizosphere of ungrafted V. vinifera var. perlette seedless planted in Sonora, Mexico using Amplified Fragment Length Polymorphism (AFLP. Results: In the selective amplification using eight primer combinations we obtained a total of 446 AFLP markers with a 100% polymorphism. Out of 41 isolates, 36 different AFLP patterns were observed and five were replicates of the same pattern. The dendrogram shows inter- and intrapopulation similarity indexes of 0.26, 0.98 and 0.31, 0.98, respectively. Six groups emerged from the principal components analysis, five of which were clearly defined, while the other one was spread out. Conclusion: We conclude that R. solani growing in Sonoran vineyards shows a high degree of genetic variability, even under similar environmental conditions.
Liang, X Y; Zhang, X Q; Bai, S Q; Huang, L K; Luo, X M; Ji, Y; Jiang, L F
Chicory is a crop with economically important roles and is cultivated worldwide. The genetic diversity and relationship of 80 accessions of chicories and endives were evaluated by sequence-related amplified polymorphism (SRAP) markers to provide a theoretical basis for future breeding programs in China. The polymorphic rate was 96.83%, and the average polymorphic information content was 0.323, suggesting the rich genetic diversity of chicory. The genetic diversity degree of chicory was higher (GS = 0.677) than that of endive (GS = 0.701). The accessions with the highest genetic diversity (effective number of alleles, NE = 1.609; Nei's genetic diversity, H = 0.372; Shannon information index, I = 0.556) were from Italy. The richest genetic diversity was revealed in a chicory line (NE = 1.478, H = 0.289, I = 0.443) among the 3 types (line, wild, and cultivar). The chicory genetic structure of 8 geographical groups showed that the genetic differentiation coefficient (GST) was 14.20% and the number of immigrants per generation (Nm) was 3.020. A GST of 6.80% and an Nm of 6.853 were obtained from different types. This observation suggests that these chicory lines, especially those from the Mediterranean region, have potential for providing rich genetic resources for further breeding programs, that the chicory genetic structure among different countries obviously differs with a certain amount of gene flow, and that SRAP markers could be applied to analyze genetic relationships and classifications of Cichorium intybus and C. endivia. PMID:25299087
Oyuna, N Yu; Moiseyeva, I G; Sevastianova, A A; Vakhrameev, A B; Alexandrov, A V; Kuzevanova, A Yu; Alimov, A A; Sulimova, G E
For the first time, the genetic diversity of the Spangled Orloff chickens was studied by analyzing the polymorphism of the hypervariable region in the D-loop of mitochondrial DNA (mtDNA). Samples for the analysis were collected at the farms ofthe All-Russia Poultry Research and Technological Institute (VNITIP), the All-Russia Institute of Farm Animal Genetics and Breeding (VNIIGRZh), and the Moscow Zoo. The D-loop partial sequences (between nucleotide positions 57 and 523) were determined according to the reference sequence of Gallus gallus spadiceus mtDNA, NC_007235 in 39 individuals obtained from these populations (GenBank Accession Nos. KM391754-KM391792). In the analyzed mtDNA fragment, a total of 20 polymorphic sites localized between positions 167 and 368, as well as at position 446, were described in Spangled Orloff chickens. One polymorphic site at position 221 (haplogroup E, haplotype ORL-2) was unique. All of the identified nucleotide changes were transition-type substitutions. Overall, based on the analysis of poly- morphic sites in the hypervariable fragment of the D-loop of Spangled Orloff chicken mtDNA, we found seven haplotypes belonging to four haplogroups (A, B, C, and E). Haplogroup E (haplotypes ORL-1, ORL-2, and ORL-3) was present in the majority of the studied individual, with the frequencies of 0.77 in the total sample and 0.47 in the VNIIGRZh farm population. Haplogroups A (haplotypes ORL-4 and ORL-7), B (ORL-6), and C (ORL-5) were found only in samples from the VNIIGRZh farm. The studied mtDNA region revealed a lower level of polymorphism in the VNITIP and Moscow Zoo populations, which only had the ORL-1 and ORL-3 haplotypes belonging to Haplogroup E, respectively. Our data suggested that the studied Spangled Orloff chicken populations differed in the composition and frequencies of mtDNA haplogroups and haplotypes. PMID:26606802
Claire L Watson
Full Text Available BACKGROUND: Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA from medieval bones and single nucleotide polymorphism (SNP typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution. METHODS AND FINDINGS: Skeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia and are dated around the medieval period (476 to 1350 A.D.. we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3. CONCLUSIONS: These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.
Micklos, David A.
This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms Ã¢ÂÂ which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, and computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrÃÂ©e to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nationÃ¢ÂÂs oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human
The aim of this study is to examine the discriminatory power of random amplified polymorphic DNA (RAPD) marker in Jatropha curcas, and to determine the effect of various dose exposures (0, 5, 10, f, 20 and 25 Kr) of gamma rays on J. curcas, at molecular level. All the ten random primers used produced reproducible polymorphic bands. PCR products of mutant genome revealed a total of 40 bands, out of which 27 were polymorphic. Polymorphism information content (PIC) values were ranged from 0.00 to 0.40 and the highest PIC value of 0.40 was observed in primer OPU-13 followed by primers OPAL-II and OPT-18 (0.30) while no PIC value were reported in primers OPH-18 and OPM-13. Jaccard's coefficient of similarity varied from 0.476 to 0.723, indicative of high level of genetic variation among the mutants studied. UPGMA cluster analysis indicated three distinct clusters, one comprising control while the second included four mutants viz., 10, 15, 25 and 20 Kr. The mutant 5 Kr remained distinct and formed third cluster indicating its higher genetic diversity from the rest of the mutants and control. The primer OPU-13 produced maximum number of bands (8) showed highest discriminatory power and PIC (0.40) by showing maximum number of polymorphic bands (5) when compared to other primers used. The study reveals that RAPD molecular markers can be used to assess polymorphism among the mutants and can be a useful tool to supplement the distinctness, uniformity and stability analysis for plant varietal identification and protection. (author)
Wang, Fuan; Lu, Chun-Hua; Liu, Xiaoqing; Freage, Lina; Willner, Itamar
The amplified, highly sensitive detection of DNA using the dendritic rolling circle amplification (RCA) is introduced. The analytical platform includes a circular DNA and a structurally tailored hairpin structure. The circular nucleic acid template includes a recognition sequence for the analyte DNA (the Tay-Sachs mutant gene), a complementary sequence to the Mg(2+)-dependent DNAzyme, and a sequence identical to the loop region of the coadded hairpin structure. The functional hairpin in the system consists of the analyte-sequence that is caged in the stem region and a single-stranded loop domain that communicates with the RCA product. The analyte activates the RCA process, leading to DNA chains consisting of the Mg(2+)-dependent DNAzyme and sequences that are complementary to the loop of the functional hairpin structure. Opening of the coadded hairpin releases the caged analyte sequence, resulting in the dendritic RCA-induced synthesis of the Mg(2+)-dependent DNAzyme units. The DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate leads to a fluorescence readout signal. The method enabled the analysis of the target DNA with a detection limit corresponding to 1 aM. By the design of two different circular DNAs that include recognition sites for two different target genes, complementary sequences for two different Mg(2+)-dependent DNAzyme sequences and two different functional hairpin structures, the dendritic RCA-stimulated multiplexed analysis of two different genes is demonstrated. The amplified dendritic RCA detection of DNA is further implemented to yield the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme as catalytic labels that provide colorimetric or chemiluminescent readout signals. PMID:24377284
Badali, Hamid; Yazdanparast, Seyed Amir; Bonifaz, Alexandro; Mousavi, Bita; de Hoog, G Sybren; Klaassen, Corné H W; Meis, Jacques F
Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC90s across all strains in increasing order posaconazole (0.25 μg/ml), itraconazole (1 μg/ml), voriconazole (4 μg/ml), terbinafine (4 μg/ml), caspofungin (8 μg/ml), anidulafungin (8 μg/ml), isavuconazole (16 μg/ml), amphotericin B (16 μg/ml), and fluconazole (32 μg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05). PMID:23463524
An effective PCR protocol for detecting the sequence related amplified polymorphism (SRAP) in rice was developed.One hundred and ten pairs of SRAP primers were used for segregation analysis in an F2 population derived from a cross between Shennong 606 and Lijiangxintuanheigu.Among the 110 primer pairs,35 pairs generated 143 polymorphic bands with an average of 4.09 polymorphic bands per primer pair,and 24 pairs (16.78%) showed the genetic distortion (P＜0.05).Of the 24 primer pairs,12 pairs deviated toward the male parent Shennong 606 and 11 pairs toward the female parent Lijiangxintuanheigu,only one toward heterozygote.It was found that the segregation distortion might be caused by the joint gametic and zygotic effects.
Elsheikha, H.M.; Schott, H. C.; Mansfield, L. S.
Sarcocystis neurona causes serious neurological disease in horses and other vertebrates in the Americas. Based on epidemiological data, this parasite has recently emerged. Here, the genetic diversity of Sarcocystis neurona was evaluated using the amplified fragment length polymorphism (AFLP) method. Fifteen S. neurona taxa from different regions collected over the last 10 years were used; six isolates were from clinically diseased horses, eight isolates were from wild-caught opossums (Didelph...
American watermelon heirlooms are diverse in their growth habits, fruit qualities and responses to biotic and abiotic stress. Wide ranging DNA marker tools resolved a narrow molecular diversity among these collections. The current research explored additional insights such as extent of diversity a...
Yuhui Li; Yingxia Yang; Jidong Liu; Xiuliang Wang; Tianxiang Gao; Delin Duan
With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.
Siemer, B.L.; Harrington, C.S.; Nielsen, E.M.;
analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human......Aims: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. Methods and Results: Field strains (n = 207) from human...... diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical...
In 1977, approximately 250 Mosquitofish (Gambusia affines) were transplanted from a relatively uncontaminated site into a small pond on the Oak Ridge Reservation that is heavily contaminated with radionuclides. DNA polymorphisms, using the RAPD technique, were examined in order to determine if any genetic differentiation had occurred between the two populations. Also, fish from another radionuclide-contaminated population (White Oak Lake) and two unrelated non-contaminated populations were also examined. The RAPD (Randomly Amplified Polymorphic DNA) technique uses the polymerase chain reaction with a short oligonucleotide primer to produce DNA fragments of various lengths. When analyzed by gel electrophoresis, these fragments form banding patterns similar to DNA fingerprints. A total of 26 primers were used to produce DNA band patterns, many of which revealed population differences. In addition several primers revealed banding patterns which differentiated between the Crystal Springs and Pond 3513 populations. Furthermore, bands found at high frequency in Pond 3513 and White Oak Lake populations were absent or present at a lower frequency in the non-contaminated populations. For some primers, the contaminated populations showed more DNA bands per individual, and fish with more bands had fewer DNA strand breaks than the fish with fewer bands. These data will be discussed with relation to biomonitoring programs and evolution of resistance to genotoxins in natural populations
Winkel, Bo G; Hollegaard, Mads Vilhelm; Olesen, Morten S; Svendsen, Jesper H; Haunsø, Stig; Hougaard, David M; Tfelt-Hansen, Jacob
The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare m...
Full Text Available We previously reported that multiply-primed rolling circle amplification (MRPCA using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.
Sauvaigo, S; Fouqué, B; Roget, A; Livache, T; BAZIN, H.; Chypre, C; Téoule, R
Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is...
Wang, Fuan; Elbaz, Johann; Willner, Itamar
The Zn(2+)-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal autocatalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10(-11) M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis. PMID:22404383
Erika Calvano Küchler
Full Text Available OBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by dna extraction. The concentration, purity and integrity of the dna were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 a/g (rs 4444903 polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215 using real-time PCR. RESULTS: There was no significant difference of dna yield (p=0.75 and purity (p=0.86 among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and real time pcr reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by real-time pcr presented c allele for irf6 gene polymorphism (homozygous: cc; heterozygous: Ct and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and pcr amplified after storage in mouthwash solution at room temperature.
Young, Charles C.; Burghoff, Robert L.; Keim, Lois G.; Minak-Bernero, Vera; Lute, James R.; Hinton, Stephen M.
This communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase chain reaction-amplifiable DNA from soil. This modification is to add polyvinylpyrrolidone to the agarose gel. The polyvinylpyrrolidone addition retards the electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids.
Roy, Laura M; Jaruga, Pawel; Wood, Thomas G; McCullough, Amanda K; Dizdaroglu, Miral; Lloyd, R Stephen
In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864-1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type beta,delta-elimination reaction but primarily yielded the beta-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and gamma-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome. PMID:17389588
McKean-Cowdin, Roberta; Barnholtz-Sloan, Jill; Inskip, Peter D; Ruder, Avima M; Butler, Maryann; Rajaraman, Preetha; Razavi, Pedram; Patoka, Joe; Wiencke, John K; Bondy, Melissa L; Wrensch, Margaret
A pooled analysis was conducted to examine the association between select variants in DNA repair genes and glioblastoma multiforme, the most common and deadliest form of adult brain tumors. Genetic data for approximately 1,000 glioblastoma multiforme cases and 2,000 controls were combined from four centers in the United States that have conducted case-control studies on adult glioblastoma multiforme, including the National Cancer Institute, the National Institute for Occupational Safety and Health, the University of Texas M. D. Anderson Cancer Center, and the University of California at San Francisco. Twelve DNA repair single-nucleotide polymorphisms were selected for investigation in the pilot collaborative project. The C allele of the PARP1 rs1136410 variant was associated with a 20% reduction in risk for glioblastoma multiforme (odds ratio(CT or CC), 0.80; 95% confidence interval, 0.67-0.95). A 44% increase in risk for glioblastoma multiforme was found for individuals homozygous for the G allele of the PRKDC rs7003908 variant (odds ratio(GG), 1.44; 95% confidence interval, 1.13-1.84); there was a statistically significant trend (P = 0.009) with increasing number of G alleles. A significant, protective effect was found when three single-nucleotide polymorphisms (ERCC2 rs13181, ERCC1 rs3212986, and GLTSCR1 rs1035938) located near each other on chromosome 19 were modeled as a haplotype. The most common haplotype (AGC) was associated with a 23% reduction in risk (P = 0.03) compared with all other haplotypes combined. Few studies have reported on the associations between variants in DNA repair genes and brain tumors, and few specifically have examined their impact on glioblastoma multiforme. Our results suggest that common variation in DNA repair genes may be associated with risk for glioblastoma multiforme. PMID:19318434
Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi Otimização da reação de amplificação aleatória do DNA polimórfico - reação em cadeia da polimerase para tipagem molecular de Salmonella enterica sorovar Typhi
Bianca Ramalho Quintaes
Full Text Available Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.A otimização da reação de RAPD para a caracterização de cepas de Salmonella enterica sorovar Typhi foi estudada com o objetivo de assegurar a reprodutibilidade e o poder discriminatório desta técnica. Oito cepas de Salmonella sorovar Typhi isoladas de algumas regiões do Brasil foram usadas para examinar os padrões de fragmentação produzidos quando foram empregadas concentrações diferentes do DNA molde, do iniciador, do MgCl2 e da enzima Taq DNA polimerase. Com a utilização de dois diferentes perfis de ciclos termais de baixa estringência, foram comparados os padrões de bandeamento obtidos. Um conjunto de dezesseis iniciadores foi avaliado quanto à capacidade de produzir elevado número de fragmentos distintos. Observou-se que variações associadas a todos os parâmetros testados modificaram os padrões de bandeamento. Para as amostras de Salmonella enterica sorovar Typhi utilizadas neste experimento, definiu-se um conjunto de condições para a reação de RAPD-PCR que resultou num método de tipagem simples, rápido e
Aabenhus, R.; On, Stephen L.W.; Siemer, Berit L.; Permin, H.; Andersen, L.P.
phenotypically indistinguishable but genetically distinct taxa (i.e., genornospecies) that may vary in pathogenicity. We examined 62 C concisus strains by amplified fragment length polymorphism (AFLP) profiling and correlated the results with clinical data. All C. concisus strains gave unique AFLP profiles, and...... numerical analysis of these data distributed the strains among four clusters. The clustering was of taxonomic significance: two clusters contained, respectively, the type strain (of oral origin) and a reference strain (from diarrhea) of each of the known genomospecies. Genomospecies 2 strains were more...
Kareem, Muhanned Abdulhassan; Abdulzahra, Ameer Ibrahim; Hameed, Imad Hadi; Jebor, Mohammed Abdullah
The aim of this research is to study the mitochondria non-coding region by using Sanger sequencing technique and establish the degree of variation characteristic of a fragment. FTA® Technology (FTA™ paper DNA extraction) is utilized to extract DNA. A portion of a non-coding region encompassing positions 438 to 574 for HVIII amplified in accordance with the Anderson reference sequence. PCR products purified by EZ-10 spin column were then sequenced and detected by using the ABI 3730xL DNA analyzer. The new polymorphic positions 469 and 482 that are described in this study may in future be suitable sources for identification purpose. The data obtained can be used to identify variable nucleotide positions characterized by frequent occurrence, most promising for identification variants. PMID:25703852
Full Text Available The uncertainty about whether, in China, the genus Melia (Meliaceae consists of one species (M. azedarach Linnaeus or two species (M. azedarach and M. toosendan Siebold & Zuccarini remains to be clarified. Although the two putative species are morphologically distinguishable, genetic evidence supporting their taxonomic separation is lacking. Here, we investigated the genetic diversity and population structure of 31 Melia populations across the natural distribution range of the genus in China. We used sequence-related amplified polymorphism (SRAP markers and obtained 257 clearly defined bands amplified by 20 primers from 461 individuals. The polymorphic loci (P varied from 35.17% to 76.55%, with an overall mean of 58.24%. Nei’s gene diversity (H ranged from 0.13 to 0.31, with an overall mean of 0.20. Shannon’s information index (I ranged from 0.18 to 0.45, with an average of 0.30. The genetic diversity of the total population (Ht and within populations (Hs was 0.37 ± 0.01 and 0.20 ± 0.01, respectively. Population differentiation was substantial (Gst = 0.45, and gene flow was low. Of the total variation, 31.41% was explained by differences among putative species, 19.17% among populations within putative species, and 49.42% within populations. Our results support the division of genus Melia into two species, which is consistent with the classification based on the morphological differentiation.
Yang, J M; Sun, G H; Zheng, X D; Ren, L H; Wang, W J; Li, G R; Sun, B C
Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P < 0.05). Most of the variance was within populations. These findings will be important for more sustainable octopus fisheries, so that this marine resource can be conserved for its long-term utilization. PMID:26634529
Morling, N; Frentz, G; Fugger, L;
We investigated the DNA restriction polymorphism (RFLP) of the Major Histocompatibility Complex (MHC) class II genes: HLA-DQA, -DQB, -DPA, and -DPB in 20 Danish patients with alopecia areata (AA) and in healthy Danes. The frequency in AA of the DQB1*0301 and DQw7 associated DQB Bgl/II 4.2 kb...... of the serologically defined HLA-DQw7 specificity. Individuals who carried both DQA1*0501 and DQB1*0301 seemed to have a further increased risk of developing AA compared to individuals carrying only one of these HLA class II genes. Analysis of the combined presence of DQB1*0301 and DPA1*0103 in AA suggests...
Luperini, B C O; Almeida, D C; Porto, M P; Marcondes, J P C; Prado, R P; Rasera, I; Oliveira, M R M; Salvadori, D M F
Obesity is characterized by increased adipose tissue mass resulting from a chronic imbalance between energy intake and expenditure. Furthermore, there is a clearly defined relationship among fat mass expansion, chronic low-grade systemic inflammation and reactive oxygen species (ROS) generation; leading to ROS-related pathological events. In the past years, genome-wide association studies have generated convincing evidence associating genetic variation at multiple regions of the genome with traits that reflect obesity. Therefore, the present study aimed to evaluate the relationships among the gene polymorphisms ghrelin (GHRL-rs26802), ghrelin receptor (GHSR-rs572169), leptin (LEP-rs7799039), leptin receptor (LEPR-rs1137101) and fat mass and obesity-associated (FTO-rs9939609) and obesity. The relationships among these gene variants and the amount of DNA damage were also investigated. Three hundred Caucasian morbidly obese and 300 eutrophic (controls) women were recruited. In summary, the results demonstrated that the frequencies of the GHRL, GHSR, LEP and LEPR polymorphisms were not different between Brazilian white morbidly obese and eutrophic women. Exceptions were the AA-FTO genotype and allele A, which were significantly more frequent in obese women than in the controls (0.23% vs. 0.10%; 0.46 vs. 0.36, respectively), and the TT-FTO genotype and the T allele, which were less frequent in morbidly obese women (p<0.01). Furthermore, significant differences in the amount of genetic lesions associated with FTO variants were observed only in obese women. In conclusion, this study demonstrated that the analyzed SNPs were not closely associated with morbid obesity, suggesting they are not the major contributors to obesity. Therefore, our data indicated that these gene variants are not good biomarkers for predicting risk susceptibility for obesity, whereas ROS generated by the inflammatory status might be one of the causes of DNA damage in obese women, favoring
Liu, Xiaoqing; Aizen, Ruth; Freeman, Ronit; Yehezkeli, Omer; Willner, Itamar
Graphene oxide (GO) is implemented as a functional matrix for developing fluorescent sensors for the amplified multiplexed detection of DNA, aptamer-substrate complexes, and for the integration of predesigned DNA constructs that activate logic gate operations. Fluorophore-labeled DNA strands acting as probes for two different DNA targets are adsorbed onto GO, leading to the quenching of the luminescence of the fluorophores. Desorption of the probes from the GO, through hybridization with the target DNAs, leads to the fluorescence of the respective label. By coupling exonuclease III, Exo III, to the system, the recycling of the target DNAs is demonstrated, and this leads to the amplified detection of the DNA targets (detection limit 5 × 10(-12) M). Similarly, adsorption of fluorophore-functionalized aptamers against thrombin or ATP onto the GO leads to the desorption of the aptamer-substrate complexes from GO and to the triggering of the luminescence corresponding to the respective fluorophore, thus, allowing the multiplexed analysis of the aptamer-substrate complexes. By designing functional fluorophore-labeled DNA constructs and their interaction with GO, in the presence (or absence) of nucleic acids, or two different substrates for aptamers, as inputs, the activation of the "OR" and "AND" logic gates is demonstrated. PMID:22404375
Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD analysis Caracterização genotípica de Haematobia irritans procedentes de diferentes regiões geográficas brasileiras baseada na análise do DNA polimórfico amplificado ao acaso (RAPD-PCR
Luciana G. Brito
Full Text Available Blood-sucking diptera are important parasites in bovine production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.Dípteras hematófagos são importantes parasitas dentro de sistemas de produção de bovinos, especialmente em confinamento. Haematobia irritans, a mosca-dos-chifres, é uma das espécies que maiores problemas causa em sistemas de produção de bovinos, dado ao intenso estresse que impõe aos animais. Um importante aspecto quando se estuda a variabilidade genética dentro das espécies é o estudo da estrutura geográfica destas populações. Buscando-se estimar a similaridade genotípica das diferentes populações brasileiras da mosca do chifre utilizou-se a técnica do DNA polimórfico amplificado ao acaso (RAPD-PCR, que mostrou-se eficiente para tal propósito. A utilização dos marcadores moleculares gerados através da técnica de RAPD-PCR tornou possível a identificação da origem geográfica das amostras das diferentes regiões geográficas brasileiras, assim como, estimar o fluxo genotípico entre as diferentes populações brasileiras da mosca-dos-chifres.
高瀬, 有加里; 藤谷, 英男; 村上, 賢; タカセ, ユカリ; フジタニ, ヒデオ; ムラカミ, マサル; Yukari, TAKASE; Hideo, Fujitani; Masaru, MURAKAMI
The Japanese silver crucian carp (so-called ginbuna, Carassius auratus langsdrofi) has two reproduction systems; one is a sexual reproduction practiced by diploid individuals and the other is gynogenetic reproduction by triploid individuals. In this study, AFLP (Amplified Fragment Length Polymorphism) analysis was carried out to isolate and characterize genomic DNA markers for triploid ginbuna, as a step toward revealing the genomic makeup and origin of triploid ginbuna. Two valuable DNA mark...
Merkelbach, S.; Gehlen, J.; Handt, S.; Füzesi, L
Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molec...
Roninson, I B; Chin, J E; Choi, K. G.; Gros, P.; Housman, D.E.; Fojo, A; Shen, D. W.; Gottesman, M M; Pastan, I
The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. It was previously demonstrated that multidrug-resistant Chinese hamster cell lines contain an amplified, transcriptionally active DNA sequence designated mdr. This report presents evidence that multidrug-resistant sublines of human KB carcinoma cells, selected for resistance to either colchicine, vinblastine, or Adriamycin (doxorubicin), d...
Spitzer, E. D.; Lasker, B A; Travis, S J; Kobayashi, G. S.; Medoff, G
We have developed an improved scheme for the classification of environmental and clinical isolates of Histoplasma capsulatum that is based on analysis of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA). Strains were initially divided into mtDNA groups according to restriction digests of whole-cell DNA and Southern hybridization with cloned mtDNA probes. Strains within a mtDNA class could be further grouped by polymorphisms in rDNA. The majority of soil and clinical isolates from the United...
Lu, Chun-Hua; Wang, Fuan; Willner, Itamar
A generic fluorescence sensing platform for analyzing DNA by the Zn(2+)-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a "helper" nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components ("helper" sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn(2+)-dependent ligation DNAzyme-driven activation of the Mg(2+)-dependent DNAzyme. According to this method, the Mg(2+)-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay-Sachs genetic disorder mutant. PMID:22612395
Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs Variação genética entre moluscos susceptíveis e não susceptíveis à infecção pelo Schistosoma através da análise do DNA polimórfico amplificado aleatóriamente (RAPDs
Abdel-Hamid Zaki ABDEL-HAMID
Full Text Available Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers were used to amplify the extracted DNA by the polymerase chain reaction (PCR followed by polyacrylamide gel electrophoresis (PAGE and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.A susceptibilidade de moluscos à infecção por certos trematódeos e a sua capacidade como hospedeiro para o contínuo desenvolvimento é o problema mais deslumbrante nas relações parasita hospedeiro. O presente trabalho, focaliza nosso interesse na genética dos moluscos para determinar quais genes ou produtos gênicos são especificamente responsáveis pela susceptibilidade do molusco à infecção. DNA de alto peso molecular, foi extraído de ambos moluscos susceptíveis e não susceptíveis da espécie Biomphalaria tenagophila. Iniciadores aleatórios com 10 pares de bases foram usados na amplificação aleatória (RAPD de ambos os DNAs e análise por eletroforese em gel de poliacrilamida e coloração com prata. Os resultados mostram que a amplificação aleatória do DNA representa um eficiente caminho para a comparação dos genomas desde que marcadores moleculares foram detectados como variantes genéticos entre os moluscos susceptíveis e não susceptíveis.
The PCR-based Escherichia coli O157 (O157) strain typing system, Polymorphic Amplified Typing Sequences (PATS), targets insertions-deletions (Indels) and single nucleotide polymorphisms (SNPs) at the XbaI and AvrII(BlnI) restriction enzyme sites, respectively, besides amplifying four known virulenc...
Yen, Hsiu-Chuan; Li, Shiue-Li; Hsu, Wei-Chien; Tang, Petrus
High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detectin...
Harper, P S; O'Brien, T.; Murray, J M; Davies, K. E.; Pearson, P; Williamson, R
Two DNA restriction fragment length polymorphisms show genetic linkage to the Duchenne muscular dystrophy locus on the short arm of the X chromosome. Examples are given of families in which these polymorphisms can be used in the prediction of genotype for this disorder.
Yoo, Hanwook; Warner, C.A.; Chen, Chiahsiang; Desnick, R.J. (Mount Sinai School of Medicine, New York, NY (United States))
Acute intermittent porphyria (AIP), an autosomal dominant inborn error of heme biosynthesis, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (HMB-synthase). Heterozygous individuals are prone to life-threatening acute neurologic attacks, which are precipitated by certain drugs and other metabolic, hormonal, and nutritional factors. Since the biochemical diagnosis of heterozygous individuals has been problematic, recent efforts have focused on the identification of mutations and diagnostically useful restriction fragment length polymorphisms (RFLPS) in the HMB-synthase gene. To facilitate these endeavors, the human HMB-synthase gene, including 1.1 kb of the 5[prime] flanking region, was isolated and completely sequenced in both orientations. The 10,024-bp gene contained 15 exons ranging in size from 39 to 438 bp and 14 introns ranging from 87 to 2913 bp. All intron/exon boundaries conformed to the GT/AG consensus rule. There were six Alu repetitive elements, one of the J and five of the Sa subfamilies. Analysis of the 1. I -kb 5[prime]flanking region revealed putative regulatory elements for the housekeeping promoter including AP1, AP4, SP1, TRE, ENH, and CAC. This region contained 10 HpaII sites and had an overall GC content of 54%. Three new polymorphic sites were identified by the single-strand conformation polymorphism (SSCP) technique, a common BsmAI site in intron 3 (3581 A/G), a common HinfI RFLP in intron 10 (7064 C/A), and a rare MnlI site in intron 14 (7998G/A). The allele frequencies of five previously known and the new polymorphic sites in a normal Caucasian population indicated that the intron 1 and intron 3 RFLPs were in linkage disequilibrium; however, the Hint I site segregated independently. 54 refs., 6 figs., 3 tabs.
Soodyall, H; Jenkins, T
Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were investigated in 95 individuals, consisting of 49 San ('Bushmen') and 46 Nama ('Hottentot') individuals from Namibia, using the restriction enzymes HpaI, BamHI, HaeII, MspI, AvaII and HincII. Six of the eleven types found in the pooled Khoisan sample are shared, albeit at varying frequencies, suggesting that both the San and Nama have evolved from a recent common ancestor. However, San and Nama groups differ appreciably, in particular, type 3-2 (3-1-1-2-2-2) was found in 7/49 Sekele and 25/46 Nama (chi 2  = 15.3, P = 9.17 x 10(-5)). In addition, type 4 makes up 42.8% of the types found in the San, and is not found in the Nama group. This suggests that the San and Nama have evolved along separate lineages, with little gene flow between them, following their proposed separation from a common Khoisan ancestor. Type 7-2 (3-1-1-1-1-2), most common in Negroid populations, is found at a higher frequency in the San (20.4%) than the Nama (6.5%), suggesting that miscegenation involving Negroid females and San males is more common than that between Negroid females and Nama men. The higher frequency of type 21-2 (2-1-1-1-2-2) in the Nama (13%) than in the San (4.1%), may be attributable to gene flow from the Dama into the Nama, consistent with the consequences of enslavement of the Dama by the Nama. PMID:1362872
We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from Spartina alterniflora and Sesbania macrocarpa root zones by bead-beating and purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 ...
Kuehni-Boghenbor, Kathrin; On, Stephen L.W.; Kokotovic, Branko; Baumgartner, Andreas; Wassenaar, Trudy M.; Wittwer, Matthias; Bissig-Choisat, Beatrice; Frey, Joachim
In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping ba...
Siemer, B.L.; Nielsen, Elsa; On, Stephan L.w.
Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish is...
Berisha, Naim; Millaku, Fadil; Gashi, Bekim; Krasniqi, Elez; Novak, Johannes
Primula veris L. (Primulaceae) is a long lived perennial and well known pharmaceutical plant, widely collected for these reasons in almost all SE Europe and particularly in Kosovo. The aim of the study is to determine molecular polymorphism of cowslip (P. veris L.) populations from Kosovo. DNA extracted from leaves were investigated in details for presence of polymorphism. RAPD analyses were conducted using 20 different short primers. Genomic DNA amplification profiles were analyzed and proc...
Miwa Sasaki; Shigeru Sakano; Naoko Okayama; Jumpei Akao; Tomohiko Hara; Yoshihisa Kawai; Chietaka Ohmi; Yuji Hinoda; Katsusuke Naito
Upper urinary tract transitional cell carcinoma (UUT-TCC) is quite an uncommon disease, and its prognosis differs among individuals irrespective of tumor stage. DNA repair gene polymorphisms are reported to result in the modulation of the repair capacity and might influence the prognosis of UUT-TCC. We examined the associations between functional polymorphisms in five DNA repair genes, and the prognosis of UUT-TCC in 103 UUT-TCC patients. Variant alleles in xeroderma pigmentosum complementati...
Full Text Available A variable domain of the mitochondrial DNA of the red-legged partridge Alectoris rufa was analysed by single-stranded DNA polymorphism (SSCP, in animals of different populations. Ten mitochondrial types were detected unevenly distributed among samples. A preserved natural population in Northern Spain, Fuentes Carrionas, showed the highest degree of polymorphism. Farm bred animals seem to be less variable and show some genotypes not usually found in the natural sites, suggesting an alien origin of many breeders.
Sander, T; Olson, S; Hall, J; Siebert, M; Grooms, K; Heisler, L; de Arruda, M; Neri, B
The removal of impurities and contaminants from PCR-amplified fragments is important for mutation detection methods which identify mutations based on shifts in electrophoretic mobility. This is particularly critical for assays and detection methods which use target DNA that is labeled prior to analysis and electrophoretic detection. We examined several procedures for purifying DNA amplified by the polymerase chain reaction (PCR) and their use in conjunction with a novel DNA scanning method, the Cleavase fragment length polymorphism (CFLP)* assay. In this study, a 480 bp DNA fragment, fluorescently labeled on the 5'-end of one strand, was amplified and subjected to various widely used purification procedures, including several commercially available clean-up kits. We demonstrate that visualization of the fluorescent label, as opposed to simple ethidium bromide staining, reveals the presence of considerable levels of labeled, truncated, amplification products. The various procedures were evaluated on the basis of their ability to remove these unwanted DNA fragments as well as on the degree to which they inhibited or promoted the CFLP reaction. Several procedures are recommended for use with CFLP analysis, including isopropanol precipitation, gel excision, and several commercially available spin columns. Concurrently, we evaluated (compared) a number of commonly used visualization platforms, including fluorescence imaging, chemiluminescence, and post-electrophoretic staining, for the ability to detect CFLP pattern changes. The advantages and disadvantages of different methods are discussed and amounts of DNA to be used for CFLP analysis on different detection platforms are recommended. PMID:10380752
Slomski, R. [Polska Akademia Nauk, Poznan (Poland). Zaklad Genetyki Czlowieka]|[Akademia Rolnicza, Poznan (Poland)]|[Laboratorium Genetyki Molekularnej, Poznan (Poland); Kwiatkowska, J.; Chlebowska, H. [Polska Akademia Nauk, Poznan (Poland). Zaklad Genetyki Czlowieka; Siemieniallo, B. [Akademia Rolnicza, Poznan (Poland); Slomska, M. [Laboratorium Genetyki Molekularnej, Poznan (Poland)
Characteristics of polymorphic sequences of DNA, especially satellite, mini satellite and micro satellite sequences are presented. Own experience from the use of multi and single locus analysis of DNA in paternity testing has been compared with the results of research in other laboratories. Critical points of both types of analysis are discussed. (author). 53 refs, 4 figs, 2 tabs.
In this study, three methods such as CTAB,SDS and Shanlichun methods were used to extract genomic DNA from the seedling of rape to find the best method. The principle, characters and application of SRAP were introduced. In order to obtain the optimal SRAP reaction system, the factors including concentrations of DNA, dNTP, etc. of reaction system were modified to better the system of rape. The result showed that the optimum concentrations were15ng DNA template, 0.2mM dNTP, 1.0μM primer and 2.0U Taq enzyme in this 25μL SRAP-PCR system.
Vargas Lúcia Rosane Bertholdo
Full Text Available The characterization of entomopathogenic microorganisms is important for the selection of more effective strains for use in integrated pest-control programs. Five Nomuraea rileyi strains (SA86101, GU87401, SR86151, CG128 and VA9101 were characterized using random amplified polymorphic DNA (RAPD analysis, virulence studies and assessment of chitinolytic and proteolytic activity. RAPD analysis divided the strains into two groups with a similarity coefficient of 0,76%, group 1 consisting of strains SA86101, GU87401 and SR86151 and group 2 of strains CG128 and VA9101. The LT50 varied from 165h with strain VA9101 to 246h with strain GU87401. Chitinolytic and proteolytic activity of the fungi after 144h growth in minimal medium were tested using colloidal chitin as substrate. All strains exhibited enzyme activity, with strain VA9101 having the highest chitinase activity (0,0040 mumol/mL/min the 40ºC and strain SA86101 the highest proteolytic activity. No relationship was found between RAPD analysis, virulence and chitinase or protease activity.
Maeda, M; Murayama, N; Ishii, H; Uryu, N; Ota, M; Tsuji, K; Inoko, H
The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing. PMID:2576477
Qin, Wei Jie; Yung, Lin Yue Lanry
Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Here we report a gold nanoparticle-based method that allows DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination. The precise quantification of single-stranded DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the presence of target...
Gerhold, R W; McDougald, L R; Beckstead, R B
A new method to amplify coccidia DNA by polymerase chain reaction (PCR) was developed by placing freeze-thawed oocysts in Ready-to-Go PCR bead tubes and using a 5-min initial heat denaturation step. Positive PCR reactions were found in 3 of 3 samples containing 20 or 50 oocysts; when ≤5 oocysts were used, 1 of 3 samples was positive. This technique shows potential for effectively and efficiently detecting and identifying oocysts from soil, feces, and other matter. PMID:25019284
Full Text Available It is well known that ionizing radiation (IR can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER. In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.
Full Text Available Sorghum genotypes currently used for grain production in the United States were developed from African landraces that were imported starting in the mid-to-late 19(th century. Farmers and plant breeders selected genotypes for grain production with reduced plant height, early flowering, increased grain yield, adaptation to drought, and improved resistance to lodging, diseases and pests. DNA polymorphisms that distinguish three historically important grain sorghum genotypes, BTx623, BTx642 and Tx7000, were characterized by genome sequencing, genotyping by sequencing, genetic mapping, and pedigree-based haplotype analysis. The distribution and density of DNA polymorphisms in the sequenced genomes varied widely, in part because the lines were derived through breeding and selection from diverse Kafir, Durra, and Caudatum race accessions. Genomic DNA spanning dw1 (SBI-09 and dw3 (SBI-07 had identical haplotypes due to selection for reduced height. Lower SNP density in genes located in pericentromeric regions compared with genes located in euchromatic regions is consistent with background selection in these regions of low recombination. SNP density was higher in euchromatic DNA and varied >100-fold in contiguous intervals that spanned up to 300 Kbp. The localized variation in DNA polymorphism density occurred throughout euchromatic regions where recombination is elevated, however, polymorphism density was not correlated with gene density or DNA methylation. Overall, sorghum chromosomes contain distal euchromatic regions characterized by extensive, localized variation in DNA polymorphism density, and large pericentromeric regions of low gene density, diversity, and recombination.
LI Yu-Feng; HUANG Qun-Ce; LIANG Yun-Zhang; YU Zeng-Liang
Germination capacity of alfalfa seeds under low energy N+ implantation manifests oscillations goingdown with dose strength. From analyzing alfalfa genome DNA under low energy N+ implantation by RAPD (RandomAmplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primersin total 100 primers, and fluorescence intensity of the identical DNA fragments amplified by RAPD is different be-tween CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N+ im-plantation manifests going up with dose strength.
Germination capacity of alfalfa seeds under low energy N+ implantation manifests oscillations going down with dose strength. From analyzing alfalfa genome DNA under low energy N+ implantation by RAPD (Random Amplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primers in total 100 primers, and fluorescence intensity of the identical DNA fragment amplified by RAPD is different between CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N+ implantation manifests going up with dose strength
Brillowska-Dabrowska, A.; Wianecka, M.; Dabrowski, Slawomir;
only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the Tsp......RI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to...... for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is...
Tvedegaard, Kristine C.; Parner, Erik; Hooper, Craig W.;
Multiplex SNP analysis on whole genome amplified DNA from archived dried bloodspots, a validation study Kristine C. Tvedegaard,1 Erik Parner,1 Craig W. Hooper,2 Jørn Atterman,1 Niels Gregersen3, Poul Thorsen,1 1Institute of Public Health, NANEA at Department of Epidemiology, University of Aarhus...... further development of allele specific primer extension (ASPE) for multiplex SNP analysis based on the Luminex 100 IS platform. It uses isobases (isoC and isoG) and the software MultiCode-PLx platform for data analysis and data handling. We validate the EraGen multicode system in two 6-plex assays used on.......3-100%, repeatability ranged from 99.2-99.7% and robustness ranged from 94.1-99.3%. CONCLUSION: The Multi-Code System is a highly sensitive and specific method for multiplex SNP analysis on WGA DNA from archived dried bloodspots....
d'Orgeix Christian A
Full Text Available Abstract Background Microsatellites, also called Simple Sequence Repeats (SSRs, repetitions of nucleotide motifs of 1-5 bases, are currently the markers of choice due to their abundant distribution in the genomes, and suitability for high-throughput analysis. A total of five different primer pairs were optimized for polymerase chain reaction (PCR to amplify microsatellite loci in total genomic DNA of bunchgrass lizards (Sceloporus slevini collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains. Findings The primers used for current investigation were originally designed for the Eastern Fence Lizard (Sceloporus undulatus. Five primer pairs were selected based on annealing temperatures for optimizing the PCR conditions to amplify with bunchgrass lizards. Different concentrations of DNA and annealing temperature were optimized. While keeping other reagents constant, a DNA concentration, 37.5 ng in the final reaction volume and PCR conditions of an initial denaturation of 94°C for five minutes, an annealing temperature of 55°C and final extension of 72°C for four minutes gave the best amplification for all the primer pairs. Conclusions Modifying the standard protocol for annealing temperatures and final extension time increases the success of cross amplification of specific microsatellite loci in the bunchgrass lizard. A loading volume of 5 ul DNA at a concentration of 10 ng/ul and a 2% agarose for gel electrophoresis were observed the best for cross amplification of selected five primer pairs on bunch grass lizard. Trial Registration The research was conducted with Arizona Game and Fish Department scientific collecting permits SP565256, SP657407 & SP749119 to Dr. Christian A d'Orgeix.
Blanchard, Kimberly A.
Single nucleotide polymorphisms (SNPs) are single base-pair variations that exist between individuals. There are approximately a million or more SNPs located throughout the genome of each individual animal. Therefore, by taking advantage of these unique polymorphisms, SNPs can be used to resolve questions of unknown parentage in the livestock industry. Currently a panel of 88 SNPs, obtained from a panel of 121 SNPs originally created by USDA-MARC, is commercially available from Fluidigm®. The...
Full Text Available Abstract Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters, but there are also interesting patterns of CpG methylation found outside of CpG islands. Results We compared DNA methylation patterns on both alleles between many pairs (and larger groups of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome.
王楷成; Linda van der Graaf; 陆承平; 黄保续; 范伟兴
The fluorescent amplified fragment length for Streptococcus suis was developed using 2 pairs of primers.51 to 98 fragments of highly polymorphic products were obtained by 2 pairs of primers in 12 Streptococcus suis isolates.The fluorescent amplified fragment length technique can be used to detect the DNA polymorphism of Streptococcus suis.Different serotypes or different isolates of the same serotype in Streptococcus suis can be distinguished by the method.It is available in identification or tracking for Streptococcus suis strains.%利用荧光标记技术,采用2对引物初步建立检测猪链球菌荧光DNA扩增片段长度多态性方法,结果表明12株猪链球菌扩增的多态性位点数从51～98条不等,该方法能够检测猪链球菌的多态性,区分不同血清型以及同一血清型不同特性的菌株,可用于菌株鉴定及流行病学研究中细菌源的追踪。
Li, Yongliang; Marion, Marie-Jeanne; Zipprich, Jennifer; Santella, Regina M.; Freyer, Greg; Brandt-Rauf, Paul W.
We have recently suggested that polymorphisms in metabolism and repair pathways may play a role in modulating the effects of exposure to the carcinogen vinyl chloride in the production of biomarkers of its mutagenic damage. The aim of the present study was to extend these observations by examining gene–environment interactions between several common polymorphisms in the DNA repair genes XRCC1 and ERCC2/XPD and vinyl chloride exposure on the production of vinyl chloride-induced biomarkers of m...
Jwu-Guh Tsay; Chishih Chu; Ya-Han Chuang; Ruey-Shyang Chen
Peronospora tabacina Adam, a downy mildew fungus, is a devastating disease of tobacco and a pathogen of import prohibition in Taiwan. For quarantine purpose, we developed a nested PCR method to detect this pathogen. With universal primer pair ITS1/ITS4, internal transcribed spacer (ITS) region of pathogen`s rDNA was amplified. Specific PCR primers (PT1/PT2) were designed based on ITS sequence and used to amplify a 493-bp rDNA fragments from P. tabacina. In order to increase the sensitivity, a...
Avrova, Anna O; Hyman, Lizbeth J.; Toth, Rachel L.; Toth, Ian K
The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (c...
XIAO-HONG ZHAO; GUANG JIA; YONG-QUAN LIU; SHAO-WEI LIU; LEI YAN; YU JIN; NIAN LIU
Objective To compare the asbestos-induced DNA damage and repair capacities of DNA damage between 104 asbestos exposed workers and 101 control workers in Qingdao City of China and to investigate the possible association between polymorphisms in codon 399 of XRCC1 and susceptibility to asbestosis. Methods DNA damage levels in peripheral bloodlymphocytes were determined by comet assay, and XRCC 1 genetic polymorphisms of DNA samples from 51 asbestosis cases and 53 non-asbestosis workers with a similar asbestos exposure history were analyzed by PCR/RFLP. Results The basal comet scores (3.95±2.95) were significantly higher in asbestos-exposed workers than in control workers (0.10±0.28). After 1 h H2O2 stimulation, DNA damage of lymphocytes exhibited different increases. After a 4 h repair period, the comet scores were 50.98±19.53 in asbestos-exposed workers and 18.32±12.04 in controls. The residual DNA damage (RD) was significantly greater (P＜0.01) in asbestos-exposed workers (35.62%) than in controls (27.75%). XRCC1 genetic polymorphism in 104 asbestos-exposed workers was not associated with increased risk of asbestosis. But compared with polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codon 399) and the DNA damage induced by asbestos, the comet scores in asbestosis cases with Gln/Gln, Gln/Arg, and Arg/Arg were 40.26±18.94, 38.03±28.22, and 32.01±11.65, respectively, which were higher than those in non-asbestosis workers with the same genotypes (25.58±11.08, 37.08±14.74, and 29.38±10.15). There were significant differences in the comet scores between asbestosis cases and non-asbestosis workers with Gln/Gln by Student's t-test (P＜0.05 or 0.01). The comet scores were higher in asbestosis workers with Gln/Gln than in those with Arg/Arg and in non-asbestosis workers exposed to asbestos, but without statistically significant difference. Conclusions Exposure to asbestos may be related to DNA damage or the capacity of cells to repair H2O2-induced
Shee, Cheng-Yap; Chong, Michelle S M; Ng, Irene; Chia, Tet-Fatt
Sequence polymorphisms of hypervariable region 1 were analyzed in 100 unrelated Singaporean Chinese. Ninety-five different haplotypes resulting from 113 variable sites were found between nucleotide positions 16045 and 16364. Single nucleotide polymorphism at nucleotide positions 16223, 16045, 16129, 16362 and 16189 was amongst the five highest frequencies observed in the sequences, whilst the most frequent haplotype was 16045-16223. Based on polymorphic sites observed at HV1, haplogroups A, F1a, M7b1, B5a and D4b were the most commonly observed clusters. The haplotype, nucleotide diversity and the average number of nucleotide differences were found to be 0.999, 0.028 and 9.082, respectively. The cytosine-stretch region located around nucleotide position 16189 was observed in 22% of this population sample. Transitions were found to be more predominant than transversions. PMID:15708338
Full Text Available The mitochondrial (mt DNA C5178A and A10398G polymorphisms have been reported to be associated with mental disorders such as bipolar disorder. However, the effects of these polymorphisms on temperament in healthy people are poorly understood. Evaluating healthy subjects can have the advantage of providing new strategies for maintaining psychological health and preventing mental illness. We examined the association between mtDNA polymorphisms and temperament in Japanese students. There was no significant difference in examined temperament when analysed by genotypes, 5178-10398 haplotypes, or sex. The subgroup analysis based on sex indicated that there was an interactive effect of the mtDNA A10398G polymorphism and sex on anxiety and obsession. This finding is preliminary and cannot exclude the possibility of false-positive due to small sample size (144 subjects and multiple statistical testing. Further studies involving a larger sample size or other ethnic groups are necessary to confirm that mtDNA A10398G polymorphism can be a genetic factor for temperament.
LIU Xin-she; CHEN Teng; LI Sheng-bin
Objective: To investigate the mitochondrial DNA sequence polymorphism in Chinese Dongxiang ethnic group and to provide basic data used in ethnic origin investigation and forensic purpose. Methods: Genomic DNA was extracted from the whole blood of 100 unrelated individuals of Chinese Dongxiang ethnic group by standard Chelex-100 method.The sequence polymorphism was determined by PCR amplification and direct sequencing. Results: Eighty-two polymorphic sites were identified in mtDNA D-loop region 16 091 - 16 418 np, and 88 haplotypes were found. The genetic diversity was calculated to be 0.996 9, and the genetic identity was 0.013 2. Conclusion: There are some particular polymorphic sites in Chinese Dongxiang ethnic group, and these sites provide an important basis to investigate the origin of Dongxiang and the relationship between Dongxiang and other ethnic groups. The result also suggested that sequence polymorphism from 16 091 -16 418 np in human mitochondrial DNA control region can be an useful tool for forensic identity.
During the process of alien germplasm introduced into wheat genome by chromosome engineering,extensive genetic variations of genome structure and gene expression in recipient could be induced.In this study,we performed GISH(genome in situ hybridization)and AFLP(amplified fragment length polymorphism) on wheat-rye chromosome transIocation lines and their parents to detect the identity in genomic structure of different translocation lines.The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation.Methylation sensitive amplification polymorphism(MSAP)analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines(CN12,20.15%;CN17,20.91%;CN18,22.42%),but the ratios of hemimethylated sites were significantly lowered(CN12,21.41%;CN17,23.43%;CN18,22.42%),whereas 16.37%were fully-methylated and 25.44%were hemimethylated in case of their wheat parent.Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent,including 13 hypermethylation patterns(33.74%),9 demethylation patterns(22.76%)and 7 uncertain patterns(4.07%).In further sequence analysis,the alterations of methylation pattern affected both repetitive DNA sequences,such as retrotransposons and tandem repetitive sequences,and low-copy DNA.
Rim Khlifi; Ahmed Rebai; Amel Hamza-Chaffai
Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair and were found to be associated with HNSCC in numerous studies. To establish our overall understanding of possible relationships between DNA repair gene polymorphisms and development of HNSCC, we surveyed the literature on epidemiological studies that assessed potential associations with HNSCC risk in terms of gene–environment interactions, genotype-induced functional defects in enzyme activity and/or protein expression, and the influence of ethnic origin on these associations.We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of HNSCC when DNA repair capacity is reduced.
Wenjing Wang; Shan Huang; Jingjing Li; Kai Rui; Jian-Rong Zhang; Jun-Jie Zhu
The strong correlation between cancer and telomerase activity has inspired the development of new strategies to evaluate telomerase activity. Here, a personal glucose meter (PGM) system that uses DNA-based machine amplification to detect telomerase in cancer cells is reported. In this assay, telomerase elongation products are amplified in the form of another type of product by a DNA-based machine. This process can only be activated by the hybridization of the extended telomerase substrate (TS...
Lieschen De Vos
Full Text Available The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.
Full Text Available The present work was made to determine the suitability of RAPD analysis for the identification of specimens of two subspecies of South American rattlesnakes (Crotalus durissus terrificus and Crotalus durissus collilineatus. The 11 arbitrary primer sequence tested amplified a total of 161 bands, of which 31% were polymorphics when compared with Crotalus specimens. The combining results from different primers allowed the identification of all the specimens under analysis. Several characteristic bands of each subspecies were identified. Dendrogram, based on RAPD markers, show three groups that corresponded to the subspecies of Crotalus, and to Bothrops jararaca specimen.O presente trabalho foi desenvolvido visando determinar a aplicabilidade da análise de RAPD na identificação de indivíduos pertencentes a duas sub-espécies de cascavéis sul-americanas (Crotalus durissus terrificus e Crotalus durissus collilineatus. As 11 sequências arbitrárias inicializadoras testadas nestes experimentos, amplificaram um total de 161 bandas, das quais 31% foram polimórficas na comparação entre espécimes de Cratalus. Os resultados combinados obtidos com as distintas sequências inicializadoras, permitiram a identificação de todos os indivíduos avaliados. Várias bandas características de cada sub-espécie foram identificadas. O dendrogama, baseado em marcaodres de RAPD, mostra tres grupos que correspondem às duas sub-espécies de Crotalus e ao especimen de Bothrops jararaca.
Merkelbach, S.; Gehlen, J.; Handt, S.; Füzesi, L.
Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9137080
It is well known that the yield of DNA recovered form tissues preserved in formalin is inversely proportional to the stored duration. How is the quality? We tested the quality of DNA from archival tissues of atomic-bomb survivors stored in formalin for decades with the parameters of gene amplification efficiency by a polymerase chain reaction. All of the DNA extracted from the tissues preserved in formalin for 30 years amplified the 54- and 61-base pairs of the DNA fragments successfully. The direct sequencing of the PCR products confirmed the accurate amplification of the target sequence. A further trial to amplify the longer sequence of 111 base pairs succeeded in 20% of the samples tested. From these results, we propose a new utility of archival samples for the analysis of single nucleotide sequence polymorphism of genes, no matter how long the samples have been preserved in formalin. (author)
Šrám, Radim; Binková, Blanka; Dejmek, Jan; Chvátalová, Irena; Solanský, I.; Topinka, Jan
Roč. 608, - (2006), s. 121-128. ISSN 1383-5718 R&D Projects: GA MŽP SL/5/160/05; GA MŽP SL/740/5/03; GA MŽP(CZ) SI/340/2/00 Institutional research plan: CEZ:AV0Z50390512 Keywords : pregnancy outcomes * birth weight * genetic polymorphism Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 2.122, year: 2006
Kolomyjec, Stephen H; Grant, Tom R; Blair, David
We identified and optimized 10 microsatellite loci for the platypus, Ornithorhynchus anatinus (Monotremata: Ornithorhynchidae), and screened 21 individuals from the southern tablelands area of New South Wales, Australia. Each polymorphic locus possessed between two and 12 alleles with observed heterozygosities between 0.118 and 0.950. The intent of this effort was to provide informative loci for studies on the population genetics of this species. PMID:21585993
Oikawa, Haruna; Tun, Zaw; Young, David R; Ozawa, Hiroyasu; Yamazaki, Kentaro; Tanaka, Einosuke; Honda, Katsuya
Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself. PMID:12237124
LUO Su; ZHANG Feng-chun; SUN Chang-jiang; LIU Cheng-bai; ZHUANG Jiang-xing; ZHANG Jin
The DNA of P3 promoter region of IGF-Ⅱ gene was obtained by means of PCR technique. The examination of DNA polymorphism by restriction endonuclease BstE Ⅱ and the examination of AFP by bioluminescence immunoassay technique were carried out. The results have a significant difference(P<0.005). But the positive rate of AFP is higher than that of DNA polymorphism. The experimental result shows that the change of the DNA polymorphism of IGF-Ⅱis not the only carcinogenic factor. The suggested unite examination is the best method for the diagnosis of the primary hepatocellular carcinoma.
The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN
Gibbs, R.A. (Baylor College of Medicine, Houston, TX (USA)); Nguyen, Phinga (Howard Hughes Medical Institute, Houston, TX (USA)); McBride, L.J.; Koepf, S.M. (Applied Biosystems, Foster City, CA (USA)); Caskey, C.T. (Baylor College of Medicine, Houston, TX (USA) Howard Hughes Medical Institute, Houston, TX (USA))
The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.
Use of data on mtDNA morph distributions from six Asian populations has shown that the observed number of different mtDNA morphs is too large when compared with the number expected on the basis of the observed gene diversity in the mtDNA genome. This excess number of morphs mainly occurs through an excess of rare morphs, and this discrepancy is more pronounced in a pooled sample of five Asian populations. It is suggested that this discrepancy is probably due to internal heterogeneity in each ...
刘晓明; 廉翠红; 金礼吉; 安利佳; 杨国玲; 林熙然
Objectives To investigate the DNA polymorphism of Sporothrix schenckii (S.schenckii) and to find the relationship between DNA patterns and geographic areas and clinical manifestations. Method The total DNA was extracted with hexadecyltrimethyl-ammonium bromide. Random A mplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S.schenckii collected from different areas and isolated from di fferent clinical types. Results Of seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5'-ACCCGACCTG-3', 5'-GAGAGCCAAC-3', 5 '-GGTGAC～GCAG-3' respectively. The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability. Differ ent isolates showed a common conserved DNA band with the same primer. Different clinical types showed different genotypes. Conclusion RAPD analysis is useful in DNA typing of S.schenckii, the DNA band type of which is related to geographic origin and Clinical manifestation.
Kamimura, K; Wakai, S; Sugio, T
The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis. PMID:11414499
Genomic DNA of 15 randomly selected unrelated animals and from two sire families (11 animals) of the Sahiwal breed of Zebu cattle were investigated. Four oligonucleotide probes - (GTG)5, (TCC)5, (GT)8 and (GT)12 - were used on genomic DNA digested with restriction enzymes AluI, HinfI, MboI, EcoRI and HaeIII in different combinations. All four probes produced multiloci fingerprints with differing levels of polymorphisms. Total bands and shared bands in the fingerprints of each individual were in the range of 2.5 to 23.0 KB. Band number ranged from 9 to 17, with 0.48 average band sharing. Probes (GT)8, (GT)12 and (TCC)5 produced fingerprinting patterns of medium to low polymorphism, whereas probe (GTG)5 produced highly polymorphic patterns. Probe (GTG)5 in combination with the HaeIII enzyme was highly polymorphic with a heterozygosity level of 0.85, followed by (GT)8, (TCC)5 and (GT)12 with heterozygosity levels of 0.70, 0.65 and 0.30, respectively. Probe GTG5 or its complementary sequence CAC5 produced highly polymorphic fingerprints, indicating that the probe can be used for analysing population structure, parentage verification and identifying loci controlling quantitative traits and fertility status. (author)
Berntsson, Shala Ghaderi; Wibom, Carl; Sjöström, Sara;
The purpose of this study was to explore the variation in DNA repair genes in adults with WHO grade II and III gliomas and their relationship to patient survival. We analysed a total of 1,458 tagging single-nucleotide polymorphisms (SNPs) that were selected to cover DNA repair genes, in 81 grade II...... different DNA repair genes (ATM, NEIL1, NEIL2, ERCC6 and RPA4) which were associated with survival. Finally, these eight genetic variants were adjusted for treatment, malignancy grade, patient age and gender, leaving one variant, rs4253079, mapped to ERCC6, with a significant association to survival (OR 0...
Hsu, Chung-Lun; Jiang, Haowei; Venkatesh, A G; Hall, Drew A
Over the past two decades, nanopores have been a promising technology for next generation deoxyribonucleic acid (DNA) sequencing. Here, we present a hybrid semi-digital transimpedance amplifier (HSD-TIA) to sense the minute current signatures introduced by single-stranded DNA (ssDNA) translocating through a nanopore, while discharging the baseline current using a semi-digital feedback loop. The amplifier achieves fast settling by adaptively tuning a DC compensation current when a step input is detected. A noise cancellation technique reduces the total input-referred current noise caused by the parasitic input capacitance. Measurement results show the performance of the amplifier with 31.6 M Ω mid-band gain, 950 kHz bandwidth, and 8.5 fA/ √Hz input-referred current noise, a 2× noise reduction due to the noise cancellation technique. The settling response is demonstrated by observing the insertion of a protein nanopore in a lipid bilayer. Using the nanopore, the HSD-TIA was able to measure ssDNA translocation events. PMID:26595927
Full Text Available The grain of Chenopodium quinoaWilld. (Quinua is important worldwide for its high nutritional value, being the principal source of proteins of the settlers of the Peruvian Bolivian altiplano. In the process of the care and maintenance of the species in the germplasm banks for quinua there have been programs developed to allow the evaluation of genetic variation to increase the quality of the grain, the resistance to diseases, and dryness tolerance and to modulate the content of saponins. A current question is to discriminate among the varieties using molecular sensitive technologies like the RAPDs, microsatellites, RFLP; our aim was to evaluate the polymorphism of six varieties of quinua using AFLP's technology (Amplified Fragment Length Polymorphism. Varieties of quinua: Quillahuaman INIA (Q, Mantaro (M, Hualhuas (H, Real Boliviana (B, Salcedo INIA(S and Illpa INIA(I, were evaluated in combinations of five pairs of primers using adapters for EcoRI and MseI to determine their polymorphisms. Our results found three combinations of major polymorphism E33/M60, E32/M48 and E32/M61, that we were able to discriminate against the variety Mantaro (M as a variety removed from the others of the southern Peruvian altiplano – Bolivian, according to UPGMAanalysis. This combination of primers also discriminate the varieties Illpa INIA and Salcedo INIA as being varieties obtained by crossings for genetic improvement.
McCleary, Tim S; Robichaud, Rodney L; Nuanes, Steve; Anagnostakis, Sandra L; Schlarbaum, Scott E; Romero-Severson, Jeanne
Hybridization between butternut (Juglans cinerea), a forest tree native to eastern North America, and Japanese walnut (J. ailantifolia), a tree tolerant to the lethal fungal disease butternut canker, casts doubt on the genetic identity of the remaining butternuts. We report a diagnostic test to distinguish the J. cinerea chloroplast from the J. ailantifolia chloroplast using cleaved amplified polymorphic sequences resolvable in 1.5% agarose gels. J. ailantifolia maternal ancestry in naturally regenerated stands provides a site selection criterion for studies of introgression dynamics when the non-native parent and the hybrids tolerate a disease to which the native species is susceptible. PMID:21564682
Guney, Ozgur; Ak, Handan; Atay, Sevcan; Ozkaya, Ali Burak; Aydin, Hikmet Hakan
The accumulation of mutations in mitochondrial DNA is a widely recognized mechanism for aging and age related diseases. However, studies indicate that some mutations could be beneficial to longevity by slowing down the function of the electron transport chain, reducing free radical production. In this study, we re-sequenced the entire mitochondrial DNA from 50 individuals and examined aging-related variations in the Turkish population. We evaluated sequence data by comparing whole SNP frequencies, individual SNP frequencies, the effect of SNPs, SNP accumulation in certain mtDNA regions and haplotype profiles between elderly and control groups. The frequency of total mitochondrial SNPs was significantly higher in nonagenarians than controls (p=0.0094). Furthermore, non-coding, synonymous and tRNA mutations were more prevalent in the 90+ group compared to controls (p=0.0001, pmutations in the D-loop region and genes encoding Complex I subunits (ND1-6) (pmutation frequency of Complex I genes in aged subjects (p<0.0001). Haplotype H was also significantly increased in the control group (p=0.0405). Overall, our findings support a role for mitochondrial genome variations and the functionality of oxidative phosphorylation in longevity. In this report, we sequenced the whole mtDNA of the Turkish population for the first time. PMID:24792352
Full Text Available Abstract Background Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods. Results Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B and Interferon-gamma receptor (IFNGR1 SNP analysis, and Interleukin-1 receptor antagonist (IL1RN variable tandem repeats (VNTR in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions. Conclusion The PCR
Full Text Available Abstract Background Recent studies have reported associations of DNA repair pathway gene variants and risk of various cancers and precancerous lesions, such as chronic atrophic gastritis (CAG. Methods A nested case-control study within the German population-based ESTHER cohort was conducted, including 533 CAG cases and 1054 controls. Polymorphisms in eleven DNA repair genes (APEX1, ERCC1, ERCC2/XPD, PARP1 and XRCC1, in CD3EAP/ASE-1 and PPP1R13L were analysed. Results No association was disclosed for any of the analysed polymorphisms. Nor did stratified analyses according to ages Conclusions The results of this large German case-control study do not reveal associations of DNA repair pathway polymorphisms and risk of CAG. On the basis of a large number of CAG cases, they do not support associations of DNA repair pathway SNPs with CAG risk, but suggest the need of larger studies to disclose or exclude potential weak associations, or of studies with full coverage of candidate genes.
Highlights: → Individuals experiencing pesticide exposure had greater mtDNA content. → OGG1 genotype may modulate mtDNA content in pesticide-exposed fruit growers. → No association between MnSOD genotypes and mtDNA content was revealed in this study. -- Abstract: Exposure to pesticides has the capacity to cause mitochondrial dysfunction. An increase mitochondrial DNA (mtDNA) content has also been suggested to relate with DNA damaging agent. In mitochondria, the manganese superoxide dismutase (MnSOD) is a scavenger of reactive oxygen species, and the 8-oxoguanine DNA glycosylase (OGG1) is the major DNA glycosylase for the repair of 8-oxoG lesions. However, the alteration of mtDNA content elicited by pesticide exposure in people with genetic variations in MnSOD or OGG1 has not been investigated. In this study, the mitochondrial to nuclear DNA ratio was quantified in the peripheral blood of 120 fruit growers who experienced pesticide exposure and 106 unexposed controls by real-time quantitative polymerase chain reaction (real-time qPCR). Questionnaires were administered to obtain demographic data and occupational history. The MnSOD and OGG1 genotypes were identified by the PCR based restriction fragment length polymorphism analysis. After adjusting for confounding effects, multiple regression model revealed that subjects experiencing high or low pesticide exposure had a greater mtDNA content than that of controls. The OGG1 Ser-Ser genotype was also associated with an increased mtDNA content. No association between MnSOD genotype and mtDNA content was revealed. Thus, subjects experiencing pesticide exposure had greater mtDNA content and the OGG1 genotype may modulate mtDNA content in pesticide-exposed fruit growers.