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Primer Pairs (primer + pair)
Kinds of Primer Pairs Terms modified by Primer Pairs Selected AbstractsIdentification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequencesMEDICAL AND VETERINARY ENTOMOLOGY, Issue 4 2008J. S. TOWNZEN Abstract Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed. [source] Characteristics of 11 polymorphic microsatellite markers in the red imported fire ant, Solenopsis invicta BurenMOLECULAR ECOLOGY RESOURCES, Issue 3 2009RAJESH B. GARLAPATI Abstract We have characterized 11 polymorphic microsatellite loci in the invasive ant Solenopsis invicta. Primer pairs were evaluated on fire ants collected from monogyne mounds in Lauderdale County, Mississippi. The observed and effective number of alleles ranged from two to six and from 1.31 to 2.64, respectively. The observed and expected heterozygosity values ranged from 0.1613 to 0.7826 and from 0.1491 to 0.6242, respectively. The polymorphism information content of the microsatellites ranged from 0.1482 to 0.6208. Probability tests indicated significant deviations from the Hardy,Weinberg equilibrium at three loci. Pairwise tests did not detect linkage disequilibrium between any pair of loci. [source] Microsatellites from kousa dogwood (Cornus kousa)MOLECULAR ECOLOGY RESOURCES, Issue 4 2008PHILLIP A. WADL Abstract Microsatellite loci were identified from Cornus kousa,National'. Primer pairs for 86 loci were developed and of these, eight were optimized and screened using genomic DNA from 22 kousa cultivars. All optimized loci were polymorphic and the number of alleles per locus ranged from three to 17. Observed heterozygosity ranged from 0 to 0.3 and expected heterozygosity ranged from 0.38 to 0.91. These microsatellites will be useful in population studies, and a breeding programme for cultivar development of Cornus species. [source] Microsatellite-enriched genomic libraries as a source of polymorphic loci for Schistosoma mansoniMOLECULAR ECOLOGY RESOURCES, Issue 2 2007N. B. RODRIGUES Abstract Microsatellite markers for Schistosoma mansoni were developed using four genomic microsatellite-enriched libraries. Microsatellites were observed in 65.4% of all sequences. Primer pairs were designed and tested for 23 loci. Eighteen loci produced amplification products, out of which 11 were polymorphic and were further characterized on 100 individuals of S. mansoni. Two to 19 alleles per locus were detected. The average values of expected and observed heterozygosities among the 11 loci were 0.79 and 0.59, respectively. [source] Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)MOLECULAR ECOLOGY RESOURCES, Issue 2 2003S. Aceto Abstract The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73. [source] Microsatellites for the Tasmanian devil (Sarcophilus laniarius)MOLECULAR ECOLOGY RESOURCES, Issue 2 2003Menna E. Jones Abstract The Tasmanian devil (Sarcophilus laniarius), a medium-sized predator/scavenger, is the largest member of the short-lived carnivorous marsupial Family Dasyuridae. Now restricted to Tasmania, populations are impacted by habitat clearance and anthropogenic mortality and genetic studies could be of value in informing levels of genetic diversity, mating system, dispersal and the effects of natural and anthropogenic landscape features on gene flow. Microsatellite markers were isolated from a partial, size-selected genomic library that was enriched for microsatellite sequences. Primer pairs were developed for 11 polymorphic dinucleotide microsatellite loci that conform with Hardy,Weinberg equilibrium and reveal moderate genetic variability across the species range. [source] Novel SSR Markers for Polymorphism Detection in Pigeonpea (Cajanus spp.)PLANT BREEDING, Issue 2 2010R. K. Saxena With 1 figure and 4 tables Abstract With an objective to expand the repertoire of molecular markers in pigeonpea (Cajanus cajan), 36 microsatellite or simple sequence repeat (SSR) loci were isolated from a SSR-enriched genomic library. Primer pairs were designed for 23 SSR loci, of which 16 yielded amplicons of expected size. Thirteen SSR markers were polymorphic amongst 32 cultivated and eight wild pigeonpea genotypes representing six Cajanus species. These markers amplified a total of 72 alleles ranging from two to eight alleles with an average of 5.5 alleles per locus. The polymorphic information content for these markers ranged from 0.05 to 0.55 with an average of 0.32 per marker. Phenetic analysis clearly distinguished all wild species genotypes from each other and from the cultivated pigeonpea genotypes. These markers should be useful for genome mapping, trait mapping, diversity studies and assessment of gene flow between populations in pigeonpea. [source] Novel microsatellite markers for the analysis of Phytophthora infestans populationsPLANT PATHOLOGY, Issue 3 2006A. K. Lees Co-dominant microsatellite molecular markers for Phytophthora infestans were developed and their potential for monitoring the genetic variation in populations was demonstrated in the UK, across Europe and worldwide. Markers were developed according to two strategies. First, several thousand P. infestans expressed sequence tag (EST) and bacterial artificial chromosome (BAC) sequences were screened for the presence of simple sequence repeat (SSR) motifs, and, of these, 100 candidate loci were selected for further investigation. Primer pairs developed to these loci were tested against a panel of 10 P. infestans isolates and approximately 10% were shown to be polymorphic and therefore appropriate for further testing. Secondly, the construction and screening of a partial genomic library resulted in the development of one additional polymorphic marker. The resulting 12 SSR markers were converted to higher-throughput fluorescence-based assays and used in combination with two previously published markers to characterize a wider collection of 90 P. infestans isolates from the UK and six other countries. Several isolates from the closely related species P. mirabilis, P. ipomoea and P. phaseoli collected from around the world were also genotyped using these markers. Amongst the 90 isolates of P. infestans examined, considerable SSR diversity was observed, with 68 different genotypes and an average of 3·9 (range 2,9) alleles per locus. When other Phytophthora species were genotyped, all loci were successfully amplified and the majority were polymorphic, indicating their transferability for the potential study of other closely related taxa. [source] Twenty-eight new microsatellite loci in chicken and their cross-species amplification in Japanese quail and helmeted guinea fowlANIMAL SCIENCE JOURNAL, Issue 4 2003Boniface Baboreka KAYANG ABSTRACT Twenty-eight original chicken microsatellite markers were isolated and characterized to determine their utility as cross-reactive markers for comparative genetic mapping in the order Galliformes. Primer pairs were typed in 12 unrelated chickens and also tested on Japanese quail and helmeted guinea fowl deoxyribonucleic acid (DNA). Polymorphism was observed in 23 (82.1%) of the markers and the average number of alleles per locus was 2.9 while the mean heterozygosity was 0.19. Eleven (39.3%) of the chicken markers cross-reacted with Japanese quail DNA and 2 (7.1%) with helmeted guinea fowl DNA. The cross-reactive markers described would serve as useful resources for comparative genetic mapping in poultry species belonging to the order Galliformes. [source] Sequence-related amplified polymorphism, an effective molecular approach for studying genetic variation in Fasciola spp. of human and animal health significanceELECTROPHORESIS, Issue 2 2009Qiao-Yan Li Abstract In the present study, a recently described molecular approach, namely sequence-related amplified polymorphism (SRAP), which preferentially amplifies ORFs, was evaluated for the studies of genetic variation among Fasciola hepatica, Fasciola gigantica and the "intermediate" Fasciola from different host species and geographical locations in mainland China. Five SRAP primer combinations were used to amplify 120 Fasciola samples after ten SRAP primer combinations were evaluated. The number of fragments amplified from Fasciola samples using each primer combination ranged from 12 to 20, with an average of 15 polymorphic bands per primer pair. Fifty-nine main polymorphic bands were observed, ranging in size from 100 to 2000,bp, and SRAP bands specific to F. hepatica or F. gigantica were observed. SRAP fragments common to F. hepatica and the "intermediate" Fasciola, or common to F. gigantica and the "intermediate" Fasciola were identified, excised and confirmed by PCR amplification of genomic DNA using primers designed based on sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair-group method with arithmetic averages clustering algorithm categorized all of the examined representative Fasciola samples into three groups, representing the F. hepatica, the "intermediate" Fasciola, or the F. gigantica. These results demonstrated the usefulness of the SRAP technique for revealing genetic variability between F. hepatica, F. gigantica and the "intermediate" Fasciola, and also provided genomic evidence for the existence of the "intermediate" Fasciola between F. hepatica and F. gigantica. This technique provides an alternative and a useful tool for the genetic characterization and studies of genetic variability in parasites. [source] Development of microsatellite markers for Pythium helicoidesFEMS MICROBIOLOGY LETTERS, Issue 1 2009Yin-Ling Abstract A strategy combining dual-suppression PCR and thermal asymmetric interlaced PCR was used to determine sequences flanking microsatellite regions in Pythium helicoides. The primer pairs were designed to amplify loci containing (AC)n, (GA)n, (AGC)n, (CAC)n(CAA)n, (TCA)n and (CTTT)n repeats from the P. helicoides nuclear genome. The PCR products of each primer pair, amplified from three representative isolates collected from different hosts and locations, were cloned and sequenced. Different degrees of polymorphism were detected among these microsatellite markers. The numbers of alleles were 6, 2, 4, 11, 4 and 4 in YL-AC, YL-AGC, YL-CAA, YL-CTTT, YL-GA and YL-TCA, respectively. Allele analysis of 30 P. helicoides isolates showed length polymorphisms in all loci, except for YL-AC, using capillary electrophoresis. Thus, we have developed a simple method for designing PCR primers to amplify microsatellite markers from P. helicoides. [source] PCR primers for identification of Sirococcus conigenus and S. tsugae, and detection of S. conigenus from symptomatic and asymptomatic red pine shootsFOREST PATHOLOGY, Issue 3 2008D. R. Smith Summary Regions of diversity in the internal transcribed spacer (ITS) sequences of Sirococcus species were exploited to design primer pairs used in a PCR-based method for the identification of the conifer shoot blight pathogen Sirococcus conigenus and the closely related fungus Sirococcus tsugae. The specificity of each primer pair for the respective fungus, detection limits and utility for detection from host material were confirmed. The S. conigenus primers were then used to detect this pathogen in tissues of symptomatic or apparently healthy red pine shoots collected at six locations in Wisconsin and Michigan and results compared with those obtained using a cultural assay. For needles, bark and wood of symptomatic shoots, the mean frequencies of detection of S. conigenus using the PCR-based methods were consistent (,7.5 out of 10) and always greater than for the cultural assay. Detection from symptomatic shoots using the cultural assay was more frequent from needles than from bark or wood. Both the PCR-based method and the cultural assay detected S. conigenus in similar frequencies from asymptomatic shoots, although less frequently than from symptomatic shoots. The efficiency of the PCR-based method and its utility for direct testing of host material should make it particularly useful in areas where multiple shoot blight pathogens are found. [source] Direct genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markersFOREST PATHOLOGY, Issue 4 2005M. Bourassa Summary Two anonymous DNA markers that are revealed by single-strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono-uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono-uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re-amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono-uredinial isolates of Mmd tested. From the overall 40 mono-uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse-grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies. Résumé Deux marqueurs génétiques anonymes, révélés par analyse SSCP (Single-Strand Conformational Polymorphism) ont été développés afin de détecter des polymorphismes génétiques chez le Melampsora medusae f. sp. deltoidae (Mmd). Dans un premier temps, des isolats mono-urédiniaux ont été obtenus, puis l'ADN a été extrait à partir des urédiniospores, les produits d'amplification RAPD (Random Amplified Polymorphic DNA) ont été générés à partir de huit de ces isolats mono-urédiniaux et les résultats d'amplification ont par la suite été séparés sur gel SSCP afin d'identifier des polymorphismes entre les isolats. Les bandes sur gel SSCP représentant des loci polymorphiques putatifs entre les isolats ont été prélevées du gel, ré-amplifiées par la technique d'amplification PCR (Polymerase Chain Reaction), clonées, puis séquencées. Pour deux fragments d'ADN séquencés sur un total de trois, une paire d'amorces a été développée afin de permettre l'amplification d'un fragment de taille adéquate pour analyse SSCP (<600 pb). Chaque paire d'amorces a produit un signal d'amplification positif pour chacun des huit isolats à l'origine de ces nouvelles amorces; les produits PCR ont ensuite été analysés par la technique SSCP. Les deux loci putatifs ont révélé des polymorphismes génétiques entre les isolats. Les deux paires d'amorces ont produit un fragment d'amplification de la taille attendue pour chacun des 32 isolats mono-urédiniaux supplémentaires testés. Des 40 isolats testés, 5 et 11 allèles ont été détectés, alors que 12 et 34 isolats se sont révélés hétérozygotes (tel qu'indiqué par la présence de plus de deux bandes sur gel SSCP) pour les loci A et B, respectivement. La spécificité des deux paires d'amorces a été testée à partir de 106 isolats fongiques appartenant à différents groupes taxonomiques, incluant d'autres rouilles, de même qu'à partir de l'ADN extrait de feuilles de peupliers cultivés en serre. Un signal d'amplification positif n'a été obtenu qu'en présence d'ADN du Mmd. Les conditions d'amplification PCR ont été optimisées pour les deux paires d'amorces développées afin de permettre le génotypage directement à partir d'urédinies individuelles prélevées sur des feuilles de peuplier infectées. La possibilité de génotyper directement des urédinies individuelles permet d'éviter l'obligation de cultiver le champignon pour génotyper les individus, ce qui représente un avantage important des marqueurs génétiques développés ici, puisqu'il devient dès lors possible de traiter un grand nombre d'échantillons lors de la réalisation d'études de populations. Zusammenfassung Zum Nachweis von Polymorphismen bei Melampsora medusae f. sp. deltoidae wurden zwei anonyme DNA Marker aus einer SSCP-Analyse entwickelt. Zunächst wurden Isolate aus einzelnen Uredinien gewonnen, die DNA wurde aus den Uredosporen extrahiert und polymorphe RAPD, Amplifikationsprodukte von acht Mono-Uredinium-Isolaten wurden auf einem SSCP-Gel getrennt, um Unterschiede zwischen ihnen nachzuweisen. Banden, die bei den acht geprüften Isolaten mögliche polymorphe Loci darstellten, wurden aus dem SSCP-Gel ausgeschnitten und mit PCR reamplifiziert, dann geklont und sequenziert. Für zwei von insgesamt drei sequenzierten DNA-Fragmenten wurde ein Primerpaar entwickelt, um ein in der Grösse für die SSCP-Analyse (<600 bp) geeignetes DNA-Fragment zu amplifizieren. Jedes Primerpaar amplifizierte bei allen acht ursprünglich für ihre Entwicklung verwendeten Isolaten ein PCR-Produkt, und diese wurden anschliessend mit SSCP analysiert. Für beide putativen Loci wurden bei den Isolaten Polymorphismen festgestellt. Die beiden Primerpaare amplifizierten ein PCR-Produkt der erwarteten Grösse bei allen 32 zusätzlich geprüften Mono-Uredinium-Isolaten des Pilzes. Bei den insgesamt 40 geprüften Mono-Uredinium-Isolaten wurden für die Loci A und B 5 bzw. 11 Allele gefunden, und 12 bzw. 34 Isolate erwiesen sich als heterozygot, was durch mehr als zwei Banden auf den SSCP-Gelen angezeigt wurde. Die Spezifität der Primerpaare wurden mit 106 Pilzisolaten aus verschiedenen Taxa geprüft, darunter andere Roste sowie DNA aus gesunden Pappelblättern aus Gewächshauskulturen. DNA-Amplifikationsprodukte der erwarteten Grösse wurden nur erhalten, wenn DNA von Melampsora medusae f. sp. deltoidae präsent war. Die PCR-Amplifikations-Bedingungen mit diesen beiden Primerpaaren wurde so optimiert, dass ein Genotyping direkt bei einzelnen von infizierten Blättern entnommenen Uredinien erfolgen kann und somit eine Pilzkultur zur Charakterisierung von Individuen entfällt. Dies ermöglicht grosse Probenzahlen in Populationsstudien. [source] Use of specific PCR-based molecular markers for discrimination, rapid analysis of purity and identification of six fragrant rice varietiesINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 10 2009Ho Ky Quang Minh Summary Fragrant rice is high in quality and hence price. Therefore, fragrant rice is often mixed with non-fragrant or low grade rice. The quality of fragrant rice depends on many factors such as variety, region in which it is cultivated, handling practices, etc. In this study, two specific primer pairs, including external sense primer, external antisense primer, internal non-fragrant sense primer and internal fragrant antisense primer were used to differentiate fragrant and non-fragrant rice based on the detection of 8-bp deletion in betaine aldehyde dehydrogenase 2 gene. The purity of the fragrant rice can be detected at 1% and 2% level of adulteration with homogeneous and heterogeneous non-fragrant rice varieties, respectively, using only one primer pair (internal non-fragrant sense primer and external antisense primer). Furthermore, six particular fragrant rice varieties were identified using specific RM 1 microsatellite markers. Further analysis should be conducted to verify the discriminating power of this method of analysis for the traceability of fragrant rice varieties. [source] Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donorsJOURNAL OF MEDICAL VIROLOGY, Issue 10 2009Alice Che-Ha Chen Abstract Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18,76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission. J. Med. Virol. 81:1792,1796, 2009. © 2009 Wiley-Liss, Inc. [source] Simultaneous detection and differentiation of human polyomaviruses JC and BK by a rapid and sensitive PCR-ELAHA assay and a survey of the JCV subtypes within an Australian populationJOURNAL OF MEDICAL VIROLOGY, Issue 3 2004David M. Whiley Abstract Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol - PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory. J. Med. Virol. 72:467,472, 2004. © 2004 Wiley-Liss, Inc. [source] Simultaneous PCR Detection of the Two Major Bacterial Pathogens of GeraniumJOURNAL OF PHYTOPATHOLOGY, Issue 2 2002D. L. GLICK Xanthomonas campestris pv. pelargonii (Xcp) and Ralstonia solanacearum (Rs) are the two most important bacterial pathogens of commercially cultivated geraniums (Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reservoirs of infections in commercial growing facilities. Our objective was to design a multiplex PCR (Polymerase Chain Reaction) assay to detect infection by either or both of these pathogens. We used a previously characterized PCR primer pair for Xcp that amplifies a region of 200 bp. In addition, we designed a new primer pair specific for Rs that amplifies a region of 822 bp. With these two primer pairs, we could detect either or both pathogens. As geranium tissue extracts frequently contain inhibitors of the PCR process, a negative PCR could result from either an accurate indication that the plant was pathogen-free or from a false negative assay. We therefore designed `amplification competence' primers, targeting a portion of the geranium 18 s rRNA gene, and generating a 494-bp amplification product that confirms amplification competence and validates a negative assay result. Thus, the triple primer pair multiplex PCR screens for the two most important bacterial pathogens of geraniums simultaneously confirms amplification competence for each geranium sample. [source] Novel polymerase chain reaction primers for the specific detection of bacterial copper P-type ATPases gene sequences in environmental isolates and metagenomic DNALETTERS IN APPLIED MICROBIOLOGY, Issue 6 2010R. De la Iglesia Abstract Aims:, In the last decades, the worldwide increase in copper wastes release by industrial activities like mining has driven environmental metal contents to toxic levels. For this reason, the study of the biological copper-resistance mechanisms in natural environments is important. Therefore, an appropriate molecular tool for the detection and tracking of copper-resistance genes was developed. Methods and Results:, In this work, we designed a PCR primer pair to specifically detect copper P-type ATPases gene sequences. These PCR primers were tested in bacterial isolates and metagenomic DNA from intertidal marine environments impacted by copper pollution. As well, T-RFLP fingerprinting of these gene sequences was used to compare the genetic composition of such genes in microbial communities, in normal and copper-polluted coastal environments. New copper P-type ATPases gene sequences were found, and a high degree of change in the genetic composition because of copper exposure was also determined. Conclusions:, This PCR based method is useful to track bacterial copper-resistance gene sequences in the environment. Significance and Impact of the Study:, This study is the first to report the design and use of a PCR primer pair as a molecular marker to track bacterial copper-resistance determinants, providing an excellent tool for long-term analysis of environmental communities exposed to metal pollution. [source] A single PCR-restriction endonuclease analysis for rapid identification of Malassezia speciesLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2000J. Guillot Aims: The present study describes a system based on PCR and restriction endonuclease analysis (REA) to distinguish the seven currently recognized Malassezia species. Methods and Results: Fifty-five representative yeast isolates were examined. A single primer pair was designed to amplify the large subunit ribosomal RNA (LSU rRNA) gene of the seven Malassezia species, and identification was achieved by digestion of the PCR products with three restriction endonucleases: BanI, HaeII and MspI. A specific restriction endonuclease analysis pattern was determined for each species investigated. Moreover, PCR-REA allowed the detection and characterization of mixtures of several Malassezia species. Conclusion: PCR-REA of only the LSU rRNA gene is a reliable and rapid method to distinguish all Malassezia species. Significance and impact of the study: PCR-REA represents a considerable saving in time over currently available identification procedures. This method should be evaluated on clinical material directly. [source] Species-specific detection of Lobaria pulmonaria (lichenized ascomycete) diaspores in litter samples trapped in snow coverMOLECULAR ECOLOGY, Issue 9 2001J.-C. Walser Abstract The foliose lichen Lobaria pulmonaria has suffered a substantial decline in central and northern Europe during the twentieth century and is now considered to be critically endangered in many European lowland regions. Based on demographic studies, it has been proposed that under the present environmental conditions and forest management regimes, dispersal of diaspores and subsequent establishment of new thalli are insufficient to maintain the remnant small lowland populations. Chances of long-term survival may therefore be reduced. The data and analytical power of these demographic studies are limited. Since lichen diaspores show very few species-specific morphological characteristics, and are therefore almost indistinguishable, the accurate assessment of diaspore flux would be a fundamental first step in better understanding the life cycle of L. pulmonaria. Here we present a new molecular approach to investigate the dispersal of L. pulmonaria diaspores in its natural environment by specifically identifying small amounts of DNA in snow litter samples at varying distances from known sources. We used a species-specific polymerase chain reaction (PCR) primer pair to amplify the ribosomal internal transcribed spacer region (ITS rDNA) and a sensitive automated PCR product detection system using fluorescent labelled primers. We detected considerable amounts of naturally dispersed diaspores, deposited as far as 50 m away from the closest potential source. Diaspores were only found in the direction of the prevailing wind. Diaspore deposition varied from 1.2 diaspores per m2 per day at 50 m distance from the source to 15 diaspores per m2 per day at 1 m distance. The method described in this paper opens up perspectives for studies of population dynamics and dispersal ecology mainly in lichenized ascomycetes but also in other organisms with small, wind-dispersed diaspores. [source] Characterization of microsatellite markers for rough fescue species (Festuca spp.)MOLECULAR ECOLOGY RESOURCES, Issue 3 2006YONG-BI FU Abstract One major challenge in genetic diversity analysis of minor grass species is the lack of informative molecular markers. A set of 210 simple sequence repeat (SSR) markers developed from wheat and barley were evaluated for their transferability to three rough fescue species [Festuca altaica Trinius, F. campestris (Rydb.) and F. hallii (Vassey) Piper]. Twelve SSR primer pairs displayed scorable polymorphism among and within the species. The number of alleles per primer pair ranged from three to 17 with an average of 8.3 for all the species and greatly varied for each species. About 82% of SSR variation resided within the species. Festuca hallii was genetically most distinct among the three species. [source] Eleven new microsatellites for hop (Humulus lupulus L.)MOLECULAR ECOLOGY RESOURCES, Issue 4 2002J. Jak Abstract We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite-enriched libraries for GAn and GTn types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2,13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs. [source] Development of novel polymerase chain reaction (PCR) based microsatellite markers in Armillaria gallica by cross-species amplification and species-specific cloningMOLECULAR ECOLOGY RESOURCES, Issue 2 2002J.-B. Lefrancois Abstract Cross-species PCR amplification of Armillaria mellea group taxa with previously reported A. ostoyae microsatellite markers, indicative of flanking sequence conservation, was exploited for the species-specific isolation of simple sequence repeat (SSR) motifs from A. gallica. Six SSR motifs were sequence characterized from cloned PCR fragments generated with primers previously developed from A. ostoyae. Five novel primer pairs, designed from motif flanking regions, allowed for improved, efficient amplification in this species. One original A. ostoyae primer pair was used directly. Polymorphims were observed at wide geographical levels only. Relative cross-species amplification intensities generally supported the currently accepted molecular phylogeny of this group. [source] Identification and characterization of microsatellites in eggplantPLANT BREEDING, Issue 3 2003T. Nunome Abstract The potential of microsatellite markers for use in genetic studies in eggplant, Solanum melongena, has been evaluated. A genomic library of eggplant was screened for GA and GT repeat motifs to isolate microsatellite clones. The frequency of each repeat motif in the eggplant genome was found to be every 3200 kb for GA repeats and every 820 kb for GT repeats. Sixty-one per cent of GT repeats were found to directly flank AT repeats. A total of 37 polymerase chain reaction (PCR) primer pairs were designed, 23 of which amplified a single product or several products. The level of microsatellite polymorphism was evaluated by using S. melongena lines and related Solanum species. Two to six alleles per primer pair were displayed in the S. melongena lines and two to 13 alleles were displayed in the Solanum relatives. Seven microsatellites showed polymorphism between parental lines of the mapping population and segregated in a codominant Mendelian manner. These microsatellite loci were distributed throughout the linkage map. [source] Identification of S -alleles using polymerase chain reaction-cleaved amplified polymorphic sequence of the S -locus receptor kinase in inbreeding lines of Brassica oleraceaPLANT BREEDING, Issue 3 2002J. I. Park Abstract Identification and DNA polymorphism of the S -locus receptor kinasegene (SRK) was analysed by pollen tube tests, polymerase chain reaction-cleaved amplified polymorphic sequence (PCR-CAPS) and nucleotide sequencing. SRK -specific primers that can distinguish class and class II S haplotypes amplified single DNA fragments of 900-1050 bp. The DNA fragments of 22 inbred lines amplified with a class SRK -specific primer pair determined seven types with HinfI and EcoRII. In addition, the DNA fragments of 17 inbred lines amplified with a class II SRK -specific primer pair determined three types with Hinf1. Nucleotide sequencing of the DNA fragments amplified from 10S haplotypes showed that exons of the 3,-end in SRK are highly conserved, and that there is much variation of the introns, which produced polymorphism of the band pattern in PCR-CAPS profiles. The S haplotypes of the plants were determined by restriction analysis of PCR products and agreed with results based on pollen tube growth tests. The PCR-CAPS analysis using specific primer pairs of SRK is considered to be useful for S allele identification in breeding programmes. [source] PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic bandPLANT PATHOLOGY, Issue 2 2003H. W. Kang A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp. carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum, betavasculorum or odoriferum, or from other Erwinia spp. or bacterial genera. The RsaI digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 102 and 1 × 103 colony forming units (CFU) mL,1 and detection sensitivity was increased to as few as 2,4 CFU mL,1 after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues. [source] A comparative assessment of molecular marker assays (AFLP, RAPD and SSR) for white yam (Dioscorea rotundata) germplasm characterizationANNALS OF APPLIED BIOLOGY, Issue 3 2003H D MIGNOUNA Summary Several DNA-based marker systems are available for genetic fingerprinting of plants but information on their relative usefulness for yam germplasm characterisation is lacking. The efficiency of RAPD, AFLP and SSR markers for the assessment of genetic relationships, and for cultivar identification and discrimination among 45 West and Central African white yam cultivars belonging to 22 morphotypes/cultivar groups was investigated. Dendrograms were produced based on band pattern scores using the UPGMA method. Results showed that each of the three techniques could unequivocably identify each cultivar, but that techniques differed in the mean number of profiles generated per primer (or primer pair) per cultivar, referred to as genotype index (GI). The order of merit based on this criterion in this study was AFLPs (GI = 2.56), SSRs (GI = 0.39) and RAPDs (GI = 0.35). Yam genotypes classified in the same cultivar group based on morphology were often genetically different, emphasising the need for molecular fingerprinting in yam germplasm characterisation. AFLPs showed the highest efficiency in detecting polymorphism and revealed genetic relationships that most closely reflected morphological classification. [source] The clinical utility of the prostate specific membrane antigen reverse-transcription/polymerase chain reaction to detect circulating prostate cells: an analysis in healthy men and womenBJU INTERNATIONAL, Issue 9 2002L. Llanes Objective,To evaluate the overall specificity of nested reverse transcriptase-polymerase chain reaction (RT-PCR) to detect prostate-specific membrane antigen (PSM) mRNA in peripheral blood samples of healthy donors. Subjects and methods,Peripheral blood samples were taken from 60 healthy blood-donors (30 men and 30 women aged < 50 years) and analysed for PSM-mRNA using nested RT-PCR (in ,hot-start' conditions and confirmed using nested EcoRI restriction enzyme). Intron-spanning primer pairs specific for human PSM were deduced from the GenBank sequence (M99487) using gene software. The outer primer pair for PSM was: fwd: 1368 5,-TCACCGGGACTCATGGGTGT-3,; reverse: 1860 5,-GCCTGAAGCAATTCCAAGTCGG-3,. Inner primer pair for PSM was: fwd: 1480 5,-AAGGAAGGGTGGAGACCTAG-3,; reverse: 5-ACTGAACTCTGGGGAAGGAC-3,. The integrity of cDNAs was checked using primer pairs specific for the housekeeping gene ,-actin. The specificity and false-positive rate were calculated assuming that the underlying prostate cancer incidence was nil. Results,The first PCR was negative for all samples (100% specificity; 0% false-positive rate). The nested PCR detected 23 positive samples (23/60, 38%) with an overall specificity of 62% (false positive rate, 38%). Conclusion,Nested RT-PCR of PSM-mRNA in peripheral blood is highly unspecific. Its clinical utility in the management of prostate cancer must be low. Further development is needed of quantitative RT-PCR, primers that identify prostatic PSM or another prostate-specific marker gene to differentiate PSM mRNA from circulating prostate cells and from non-prostatic tissues. [source] DNA sequence analysis of interlocus recombination between the human T-cell receptor gamma variable (GV) and beta diversity-joining (BD/BJ) sequences on chromosome 7 (inversion 7)ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2002Scott W. Ballinger Abstract V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase. Environ. Mol. Mutagen. 40:85,92, 2002. © 2002 Wiley-Liss, Inc. [source] RAPID ADAPTIVE DIVERGENCE IN NEW WORLD ACHILLEA, AN AUTOPOLYPLOID COMPLEX OF ECOLOGICAL RACESEVOLUTION, Issue 3 2008Justin Ramsey Adaptive evolution is often associated with speciation. In plants, however, ecotypic differentiation is common within widespread species, suggesting that climatic and edaphic specialization can outpace cladogenesis and the evolution of postzygotic reproductive isolation. We used cpDNA sequence (5 noncoding regions, 3.5 kb) and amplified fragment length polymorphisms (AFLPs: 4 primer pairs, 1013 loci) to evaluate the history of ecological differentiation in the North American Achillea millefolium, an autopolyploid complex of "ecological races" exhibiting morphological, physiological, and life-history adaptations to diverse environments. Phylogenetic analyses reveal North American A. millefolium to be a monophyletic group distinct from its European and Asian relatives. Based on patterns of sequence divergence, as well as fossil and paleoecological data, colonization of North America appears to have occurred via the Bering Land Bridge during the Pleistocene (1.8 MYA to 11,500 years ago). Population genetic analyses indicate negligible structure within North American A. millefolium associated with varietal identity, geographic distribution, or ploidy level. North American populations, moreover, exhibit the signature of demographic expansion. These results affirm the "ecotype" concept of the North American Achillea advocated by classical research and demonstrate the rapid rate of ecological differentiation that sometimes occurs in plants. [source] | ||||||||||||||||||||||||||||||||||||||||