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PCR Primers (pcr + primer)
Kinds of PCR Primers Terms modified by PCR Primers Selected AbstractsComplementary DNA sequence of the HLA-B*3924 allele,INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 1 2003E. Estefanía Summary We have isolated the complete coding region of HLA-B*39 from a Spanish Caucasoid, using a new PCR primer for its 5, untranslated region. The cDNA matched partial genomic sequences of B*3924, an allele whose distribution appears to be restricted to Mediterranean and Arabian Caucasoids. A single amino acid change exclusive to B*3924 (threonine-98) distinguishes it from B*3903. [source] Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4AJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2001Kimberly B. Bleicher Abstract Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan®) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan® probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3, end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:133,142, 2001 [source] Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assaysPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 1 2009Séverine Fontaine Abstract BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations. Copyright © 2008 Society of Chemical Industry [source] Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markersANNALS OF APPLIED BIOLOGY, Issue 3 2010A. Castillo A selection of 147 wheat D-genome and 130 barley genomic simple sequence repeat (gSSR) markers were screened for their utility in Hordeum chilense, as an alien donor genome for cereal breeding. Fifty-eight wheat D-genome and 71 barley PCR primer pairs consistently amplified products from H. chilense. Nineteen wheat D-genome and 20 barley gSSR markers were polymorphic and allowed wide genome coverage of the H. chilense genome. Twenty-three of the wheat D-genome and 11 barley PCR primer pairs were suitable for studying the introgressions of H. chilense into wheat, amplifying H. chilense products of distinct size. In 88% of the markers tested, H. chilense products were maintained in the expected homeologous linkage group, as revealed by the analysis of wheat/H. chilense addition lines. Twenty-nine microsatellite markers (eight gSSRs and 21 expressed sequence tags-SSRs) uniformly distributed across the genome were tested for their utility in genetic diversity analysis within the species. Three genetic clusters are reported, in accordance with previous morphological and amplified fragment length polymorphism data. These results show that it is possible to discriminate the three previously established germplasm groups with microsatellite markers. The reported markers represent a valuable resource for the genetic characterisation of H. chilense, for the analysis of its genetic variability, and as a tool for wheat introgression. This is the first intraspecific study in a collection of H. chilense germplasm using microsatellite markers. [source] Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencingELECTROPHORESIS, Issue 12 2007Pengfeng Xiao Dr. Abstract A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1,M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10,,L of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis. [source] Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic OceanENVIRONMENTAL MICROBIOLOGY, Issue 5 2010Liping Wang Summary Alkane hydroxylases, including the integral-membrane non-haem iron monooxygenase (AlkB) and cytochrome P450 CYP153 family, are key enzymes in bacterial alkane oxidation. Although both genes have been detected in a number of bacteria and environments, knowledge about the diversity of these genes in marine alkane-degrading bacteria is still limited, especially in pelagic areas. In this report, 177 bacterial isolates, comprising 43 genera, were obtained from 18 oil-degrading consortia enriched from surface seawater samples collected from the Atlantic Ocean. Many isolates were confirmed to be the first oil-degraders in their affiliated genera including Brachybacterium, Idiomarina, Leifsonia, Martelella, Kordiimonas, Parvibaculum and Tistrella. Using degenerate PCR primers, alkB and CYP153A P450 genes were surveyed in these bacteria. In total, 82 P450 and 52 alkB gene fragments were obtained from 80 of the isolates. These isolates mainly belonged to Alcanivorax, Bacillus, Erythrobacter, Martelella, Parvibaculum and Salinisphaera, some of which were reported, for the first time, to encode alkane hydroxylases. Phylogenetic analysis showed that both genes were quite diverse and formed several clusters, most of which were generated from various Alcanivorax bacteria. Noticeably, some sequences, such as those from the Salinisphaera genus, were grouped into a distantly related novel cluster. Inspection of the linkage between gene and host revealed that alkB and P450 tend to coexist in Alcanivorax and Salinisphaera, while in all isolates of Parvibaculum, only P450 genes were found, but of multiple homologues. Multiple homologues of alkB mostly cooccurred in Alcanivorax isolates. Conversely, distantly related isolates contained similar or even identical sequences. In summary, various oil-degrading bacteria, which harboured diverse P450 and alkB genes, were found in the surface water of Atlantic Ocean. Our results help to show the diversity of P450 and alkB genes in prokaryotes, and to portray the geographic distribution of oil-degrading bacteria in marine environments. [source] Phylogenetic analysis of actinobacterial populations associated with Antarctic Dry Valley mineral soilsENVIRONMENTAL MICROBIOLOGY, Issue 3 2009Olubukola O. Babalola Summary Despite the apparent severity of the environmental conditions in the McMurdo Dry Valleys, Eastern Antarctica, recent phylogenetic studies conducted on mineral soil samples have revealed the presence of a wide diversity of microorganisms, with actinobacteria representing one of the largest phylotypic groups. Previous metagenomic studies have shown that the majority of Antarctic actinobacterial populations are classified as ,uncultured'. In this study, we assessed the diversity of actinobacteria in Antarctic cold desert soils by complementing traditional culture-based techniques with a metagenomic study. Phylogenetic analysis of clones generated with actinobacterium- and streptomycete-specific PCR primers revealed that the majority of the phylotypes were most closely related to uncultured Pseudonocardia and Nocardioides species. Phylotypes most closely related to a number of rarer actinobacteria genera, including Geodermatophilus, Modestobacter and Sporichthya, were also identified. While complementary culture-dependent studies isolated a number of Nocardia and Pseudonocardia species, the majority of the cultured isolates (> 80%) were Streptomyces species , although phylotypes affiliated to the genus Streptomyces were detected at a low frequency in the metagenomic study. This study confirms that Antarctic Dry Valley desert soil harbours highly diverse actinobacterial communities and suggests that many of the phylotypes identified may represent novel, uncultured species. [source] Diagnosis and detection of host-specific forms of Fusarium oxysporum,EPPO BULLETIN, Issue 3-4 2000R. P. Baayen Diagnosis and detection of host-specific forms of Fusarium oxysporum are traditionally based on the combination of diagnostic symptoms on the host with the presence of the fungus in the affected tissues. The classical approach is becoming increasingly problematic because more than one forma specialis may occur on a given host, along with non-pathogenic strains which are common soil and rhizosphere inhabitants. Neither formae speciales nor pathogenic races within formae speciales can be distinguished morphologically. Although united by joint pathogenicity to a given host, strains belonging to the same forma specialis need not be phylogenetically related. Development of diagnostics for host-specific groups in F. oxysporum requires monophyletic target groups. Recent studies on gene-genealogy and AFLP-based phylogenies show that the majority of formae speciales in F. oxysporum are polyphyletic (unnatural) and do not offer any prospects for the development of molecular diagnostics. In contrast, highly specific PCR primers have been developed for formae speciales (or races) that consist of a single clonal lineage, and for monophyletic groups of lineages within a forma specialis. Among others, specific PCR primers have thus been developed for F. oxysporum f. sp. basilici, specific races in F. oxysporum ff. spp. dianthi and gladioli, and for the EPPO A2 (EU II/A1) quarantine fungus F. oxysporum f. sp. albedinis which can reliably replace conventional isolation and pathogenicity testing procedures. [source] Genetic diversity and distribution of periphytic Synechococcus spp. in biofilms and picoplankton of Lake ConstanceFEMS MICROBIOLOGY ECOLOGY, Issue 2 2004Sven Becker Abstract In various water depths of the littoral zone of Lake Constance (Bodensee) cyanobacteria of the Synechococcus -type were isolated from biofilms (periphyton) on three natural substrates and an artificial one (unglazed tiles). From one tile three strains of phycoerythrin (PE)-rich Synechococcus spp. were isolated, the first examples of these organisms in the epibenthos. Phylogenetic inference based on the 16S,23S rRNA intergenic spacer (ITS-1) assigned all periphytic isolates to two clusters of the picophytoplankton clade (evolutionary lineage VI of cyanobacteria). The sequence divergence in the ITS-1 was used to design specific PCR primers to allow direct, culture-independent detection and quantification of isolated Synechococcus strains in natural periphytic and pelagic samples. Denaturing gradient gel electrophoresis (DGGE) analysis revealed depth-related differences of Synechococcus spp. distribution on tiles placed in the littoral zone. Synechococcus genotypes were observed which occurred in both the periphyton (on tiles) and in the pelagic picoplankton. A strain with one of these genotypes, Synechococcus sp. BO 8805, was isolated from the pelagic zone in 1988. Its genotype was found on tiles that had been exposed at different water depths in the littoral zone in spring and autumn of the year 2000. Quantitative analysis with a genotype-specific TaqMan probe and real-time Taq nuclease assays (TNA) confirmed its presence in the pelagic zone, although appearance of this and related genotypes was highly irregular and exhibited strong differences between consecutive years. Our results show that the ability to form significant subpopulations in pelagic and periphytic communities exists in three out of four phylogenetic clusters of Synechococcus spp. in Lake Constance. This versatility may be a key feature in the ubiquity of the evolutionary lineage VI of cyanobacteria. [source] Effects of transgenic glufosinate-tolerant oilseed rape (Brassica napus) and the associated herbicide application on eubacterial and Pseudomonas communities in the rhizosphereFEMS MICROBIOLOGY ECOLOGY, Issue 3 2002Stephen Gyamfi Abstract A containment experiment was carried out in order to evaluate possible shifts in eubacterial and Pseudomonas rhizosphere community structures due to the release of genetically modified Basta-tolerant oilseed rape and the associated herbicide application. Treatments included cultivation of the transgenic plant as well as of the wild-type cultivar in combination with mechanical removal of weeds and the application of the herbicides Basta (active ingredient: glufosinate) and Butisan S (active ingredient: metazachlor). Rhizosphere soil was sampled from early and late flowering plants as well as from senescent plants. A culture-independent approach was chosen to characterize microbial communities based on denaturing gradient gel electrophoresis of 16S rRNA gene fragments amplified from rhizosphere DNA using eubacterial and Pseudomonas -specific PCR primers. Dominant pseudomonads in the rhizosphere were analyzed by sequence analysis. Whole community and Pseudomonas electrophoresis fingerprints revealed slightly altered microbial communities in the rhizosphere of transgenic plants; however, effects were minor as compared to the plant developmental stage-dependent shifts. Both herbicides caused transient changes in the eubacterial and Pseudomonas population structure, whereas differences due to the genetic modification were still detected at the senescent growth stage. The observed differences between transgenic and wild-type lines were most likely due to unintentionally modified plant characteristics such as altered root exudation. [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] Identification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species-specific PCR primers from the ribosomal ITS regionFOREST PATHOLOGY, Issue 3 2005E. Stenström Summary Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species-specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries. Résumé Lophodermium seditiosum est un pathogène important des aiguilles sur pins, particulièrement en pépinières, et il serait nécessaire de détecter le pathogène dans sa phase latente. Les régions ITS de L. seditiosum et L. pinastri ont été amplifiées avec des amorces universelles et séquencées. La comparaison de la séquence des deux espèces a permis de développer des amorces spécifiques pour chaque espèce dans la région ITS. Les amorces ont une longueur de 18 à 24 paires de bases avec un minimum de 3 paires de bases de différence entre espèces. Ces amorces n'ont produit aucune amplification avec l'ADN des autres espèces de champignons testées ou les aiguilles saines de Pinus sylvestris. Il a également été possible de détecter L. seditiosum ou L. pinastri avec ces amorces dans des aiguilles infectées avec ou sans signe d'infection. Cette méthode s'avère très utile pour la détection d'infections latentes de L. seditiosum dans les aiguilles de P. sylvestris en pépinières. Zusammenfassung Lophodermium seditiosum ist ein starkes Nadelpathogen an Kiefern, speziell in Baumschulen. Für den Einsatz von Bekämpfungsmassnahmen wäre es von Vorteil, wenn man das Pathogen bereits während der Latenzperiode nachweisen könnte. Die ITS Regionen der ribosomalen DNA von L. seditiosum und L. pinastri wurden mit Standardprimern amplifiziert und sequenziert. Vergleiche der Sequenzen der beiden Arten erlaubten die Entwicklung von artspezifischen Primern für die ITS Regionen. Die Primerpaare waren zwischen 18 and 24 Basenpaaren lang und wiesen einen Unterschied von mindestens drei Nukleotiden auf. Die DNA von allen anderen untersuchten Pilzarten und von gesunden Pinus sylvestris Nadeln liessen sich mit keinem dieser Primerpaare amplifiziern. Lophodermium seditiosum und L. pinastri konnten mit den Primerpaaren in infizierten Nadeln mit und ohne Symptome direkt nachgewiesen werden. Die Methode eignete sich vorzüglich zum Nachweis von latenten Infektionen von L. seditiosum in P. sylvestris Nadeln in Baumschulen. [source] Development of species-specific PCR primers on rDNA for the identification of European Armillaria speciesFOREST PATHOLOGY, Issue 5 2003G. Sicoli Summary Attempts to design species-specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa. Résumé Des essais ont été réalisés pour obtenir des amorces PCR spécifiques de 6 espèces européennes d'Armillaria dans les gènes de l'ARNr. Les amorces ont été développées sur la base de la variabilité de séquence nucléotidique dans les ITS et IGS (IGS1) de l'ADN ribosomal. Quatre couples d'amorces ont permis d'obtenir des produits PCR spécifiques pour A. tabescens, A. mellea et A. ostoyae. Cependant, compte tenu des très fortes similarités de séquence entre A. borealis et A. ostoyae, et entre A. cepistipes et A. gallica, il n'a pas été obtenu d'amplification spécifique pour ces taxons. Zusammenfassung Es wird über Versuche berichtet, artspezifische Primer für sechs europäische Armillariaarten in der Region der ribosomalen RNA-Gene zu entwickeln. Als Grundlage dafür diente die Variabilität der Nukleotidsequenzen der ITS- und der IGS 1-Region der ribosomalen DNA. Vier Primerpaare ergaben spezifische PCR-Produkte für A. tabescens, A. mellea und A. ostoyae. Dagegen wurden aufgrund der grossen Ähnlichkeit der Sequenzen von A. borealis und A. ostoyae sowie von A. cepistipes und A. gallica für diese Taxa keine artspezifischen Amplifikationsprodukte erhalten. [source] Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of lynch syndrome patients,HUMAN MUTATION, Issue 5 2010Heleen M. van der Klift Abstract Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3, end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2 -specific PCR primers and MLPA probes, designed on PSVs, in the 3, duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods. Hum Mutat 31:578,587, 2010. © 2010 Wiley-Liss, Inc. [source] Use of a PCR-based approach for sequencing whole mitochondrial genomes of insects: two examples (cockroach and dragonfly) based on the method developed for decapod crustaceansINSECT MOLECULAR BIOLOGY, Issue 4 2004M. M. Yamauchi Abstract Recent development of a PCR-based approach for sequencing vertebrate mitochondrial genomes has attracted much attention as being more rapid and economical than traditional methods using cloned mtDNA and primer walking. Such a method has not been available for insect mitochondrial genomes, despite widespread use of them for the molecular phylogenetic, biogeographical and population genetic markers. A recently developed PCR-based approach for sequencing whole mitochondrial genomes of decapod crustaceans, which included the design of many versatile PCR primers for the latter, was applied with the same primers sets to mitochondrial genomes of two insects, smokybrown cockroach Periplaneta fuliginosa (Serville, 1839) and skimmer dragonfly Orthetrum triangulare melania (Selys, 1883). Almost the entire region of the two mitochondrial genomes was successfully sequenced. Features of the two mitochondrial genomes are described and the usefulness of this PCR-based approach for sequencing insect mitochondrial genomes demonstrated. [source] Identification and analysis of Lydia, a LTR retrotransposon from Lymantria disparINSECT MOLECULAR BIOLOGY, Issue 4 2000T. A. Pfeifer Abstract Degenerative PCR primers to conserved amino acid motifs were used to identify an LTR retrotransposon from Lymantria dispar. The isolated retrotransposon, Lydia, is 6655 base pairs (bp) in length and contains perfect 300 bp terminal repeats. The identified gag and pol related ORFs have a high degree of similarity to the corresponding regions of the retrotransposon Ted from Trichoplusia ni, although several reading frameshifts and missense mutations are evident. The high degree of similarity between Lydia and Ted LTRs lends support for a family of lepidopteran retrotransposons. Southern blot analysis of individuals from two geographically distinct gypsy moth populations demonstrates that Lydia is found in both populations and the position of this element within the genome of these isolated populations is variable. [source] Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experienceJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2003C.S. Harrington Abstract Aims: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. Methods and Results: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98,100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. Conclusions:Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. Significance and Impact of the Study: This work should facilitate progress towards inter-laboratory standardization of fla typing. [source] Direct analysis of sulfate reducing bacterial communities in gas hydrate-impacted marine sediments by PCR,DGGEJOURNAL OF BASIC MICROBIOLOGY, Issue S1 2009Christopher E. Bagwell Abstract Molecular investigations of the sulfate reducing bacteria that target the dissimilatory sulfite-reductase subunit A gene (dsr A) are plagued by the nonspecific performance of conventional PCR primers. Here we describe the incorporation of the FailSafeÔ PCR System to optimize environmental analysis of dsr A by PCR amplification and denaturing gradient gel electrophoresis. PCR,DGGE analysis of dsr A composition revealed that SRB diversity was greater and more variable throughout the vertical profile of a marine sediment core obtained from a gas hydrate site (GC234) in the Gulf of Mexico than in a sediment core collected from a nearby site devoid of gas hydrates (NBP). Depth profiled dsr B abundance corresponded with sulfate reduction rates at both sites, though measurements were higher at GC234. This study exemplifies the numerical and functional importance of sulfate reducing bacteria in deep-sea sedimentary environments, and incremental methodological advancements, as described herein, will continue to streamline the analysis of sulfate reducer communities in situ. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB regionJOURNAL OF FISH DISEASES, Issue 7 2007S Izumi Abstract Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA. [source] The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains§JOURNAL OF FORENSIC SCIENCES, Issue 2 2006Kerry L. Opel M.A. ABSTRACT: A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50,280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex® 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex® 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains. [source] Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Joseph Y. Fu Abstract Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of 20 volume% of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acid adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20,mM HEPES, 240,mM NaCl, pH,7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations. Copyright © 2006 John Wiley & Sons, Ltd. [source] STRUCTURAL FEATURES OF NUCLEAR GENES IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 5 2000E. Virginia Armbrust Thalassiosira weissflogii (Grun.) Fryxell et Hasle is one of the more commonly studied centric diatoms, and yet molecular studies of this organism are still in their infancy. The ability to identify open reading frames and thus distinguish between introns and exons, coding and noncoding sequence is essential to move from nuclear DNA sequences to predicted amino acid sequences. To facilitate the identification of open reading frames in T. weissflogii, two newly identified nuclear genes encoding ,-tubulin and t -complex polypeptide (TCP)-,, along with six previously published nuclear DNA sequences, were examined for general structural features. The coding region of the nuclear open reading frames had a G + C content of about 49% and could readily be distinguished from noncoding sequence due to a significant difference in G + C content. The introns were uniformly small, about 100 base pairs in size. Furthermore, the 5, and 3, splice sites of introns displayed the canonical GT/AG sequence, further facilitating recognition of noncoding regions. Six of the nuclear open reading frames displayed relatively little bias in the use of synonymous codons, as exemplified by the cDNAs encoding ,-tubulin and TCP-,. Two open reading frames displayed strong bias in the use of particular codons (although the codons used were different), as exemplified by the cDNA encoding fucoxanthin chlorophyll a/c binding protein. Knowledge of codon bias should facilitate, for example, design of degenerate PCR primers and potential heterologous reporter gene constructs. [source] HCV-RNA In Sural Nerve From Hcv Infected Patients With Peripheral NeuropathyJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001L De Martino Objective: Evaluation of hepatitis C virus (HCV) by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral nerve tissues from HCV infected patients with peripheral neuropathy. METHODS: RT-PCR was performed on homogenates of nerve biopsies from 17 consecutive HCV-positive patients with peripheral neuropathy, with or without mixed cryoglobulinemia, hospitalised from 1996 to 2000. Sural nerve specimens were frozen in iso-pentane pre-cooled in liquid nitrogen and stored at ,80°C until use. RNA was extracted from ten 7-,m thick cryostatic sections or from a nerve trunk specimen of about 3 mm length, collected from each biopsy. Three different protocols of RNA extraction were tested (1,3). Complementary DNAs (cDNAs) were obtained without or with RNasin (Promega, Madison, WI) addition in the reaction mixture to inhibit residual RNase activity. Two sets of commercially available PCR primers for the outer and the nested reaction were used. PCR products were analysed by agarose gel electrophoresis and ethidium bromide staining. Serum samples and liver specimens from proven HCV positive patients served as positive controls, whereas sera from healthy subjects were negative controls. RESULTS: Sufficient amount of RNA could be obtained either by cryostatic sections or by in toto nerve specimens. Extraction by Trizol (Gibco-BRL) allowed the best concentration and purity of RNA as assessed by biophotometry. The presence of RNasin didn't improve the cDNA synthesis. The resulting amplification product of the nested PCR was 187 bp long. We have always observed this product in our positive controls and never in the negative. Six samples from patients either with or without cryoglobulinemia resulted positive; 7 were negative. Four samples gave variable results. CONCLUSIONS: While 40% of the nerves in our series were undoubtedly HCV positive, the cause(s) of negative and variable results in the remaining samples is likely more complex than variations in the detection protocols and deserve further investigations. REFERENCES: 1) Chomczynski P, Sacchi N (1987). Anal Biochem 162:156. 2) Marquardt O et al. (1996). Med Microbiol Lett 5:55. 3) Chomczynski P (1993). Bio/Techniques 15:532. [source] Use of specific PCR primers to identify three important industrial species of Saccharomyces genus: Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianusLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2010G.V. De Melo Pereira Abstract Aim:, To develop species-specific primers capable of distinguishing between three important yeast species in alcoholic fermentation: Saccharomyces bayanus, Saccharomyces cerevisiae and Saccharomyces pastorianus. Methods and Results:, Two sets of primers with sequences complementary to the HO genes from Saccharomyces sensu stricto species were used. The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. The second pair of primers (LgHO) was also constructed, within the HO gene, composed of perfectly conserved sequences common for S. bayanus species, which generate amplicon with 700 bp. No amplification product was observed in the DNA samples from non- Saccharomyces yeasts. Saccharomyces species have also been characterized via electrophoretic karyotyping using pulsed-field gel electrophoresis to demonstrate chromosomal polymorphisms and to determine the evolutionary distances between these species. Conclusions:, We conclude that our novel species-specific primers could be used to rapidly and accurately identify of the Saccharomyces species most commonly involved in fermentation processes using a PCR-based assay. Significance and Impact of the Study:, The method may be used for routine identification of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes in less than 3 h. [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] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009A. Grover Abstract Aims:, To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification,PCR amplification). Methods and Results:, MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (, 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml,1) of bacteria within 8 h including DNA isolation. Conclusion:, MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato. Significance and Impact of study:, The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes. [source] Survival and retention of the probiotic Lactobacillus casei LAFTI® L26 in the gastrointestinal tract of the mouseLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2007P. Su Abstract Aims:, This study aimed to develop methods for the detection of the probiotic Lactobacillus casei LAFTI® L26 (L26) from mouse faeces, and to determine the survival and retention time of L26 in the mouse gastrointestinal tract. Methods and Results:, A selective medium, de Man Rogosa Sharpe (MRS) + bromocresol green + vancomycin (MGV), was designed for the isolation and enumeration of L26 from faecal samples of mice. PCR primers were designed to confirm the identity of L26-like colonies on MGV. These primers did not produce PCR products from related organisms that grew on MGV. Following the administration of L26 to BALB/c mice, faecal samples were collected and analysed using the designed methods. Survival studies showed viable L26 cells to be present in the faeces of mice for >48 h. Conclusions:, Our results suggest that L26 is able to survive and be retained within the digestive tract of mice for at least 48 h following oral administration. Significance and Impact of the Study:, MGV allows effective recovery of L26 from the background microbiota, including lactobacilli of mice. PCR was used to confirm that L26-like colonies were correctly identified as L26. Given the long retention time of L26 in the gastrointestinal tract of mice, it would appear that this probiotic strain may survive in the human gastrointestinal tract. [source] Evaluation of PCR primers from putative transcriptional regulator genes for identification of Staphylococcus aureusLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2005D. Liu Abstract Aims:, To examine if PCR primers derived from putative transcriptional regulator genes can be useful for Staphylococcus aureus identification. Methods and Results:,Staphylococcus aureus gene sequences that encode transcriptional regulators were retrieved from GenBank and compared with other DNA sequences via BLAST searches. Two uniquely present, putative transcriptional regulator genes (i.e. Sa0836 and Sa0856) were selected as a consequence and PCR primers (Sa0836F/R and Sa0856F/R) were then designed from these genes for evaluation. A total of 84 bacterial strains/isolates including 23 Staph. aureus, 18 nonaureus Staphylococcus and 43 other common bacterial isolates were examined. The results indicated that PCR primers from Sa0836 and Sa0856 recognized genomic DNA from Staph. aureus only, but not from other non-aureus Staphylococcus or common bacteria. Conclusions:, PCR detection of the putative transcriptional regulator genes Sa0836 and Sa0856 represents a useful means of identifying Staph. aureus from other bacteria. Significance and Impact of the Study:, The existence of species,species transcriptional regulator genes may be a common phenomenon in bacteria. Besides their value as novel diagnostic markers, further investigation on the putative transcriptional regulator genes Sa0836 and Sa0856 and their related products may shed light on the molecular mechanisms of Staph. aureus adaptation and virulence. [source] Development of 16S rDNA-based PCR assay for detecting Centipeda periodontii and Selenomonas sputigenaLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2000S. Sawada To detect oral motile bacteria directly from dental plaque, specific PCR primers for Centipeda periodontii and Selenomonas sputigena were designed based on the sequence analysis of their 16S rDNA. The primers were specific and sensitive enough to amplify DNA fragments from the available oral bacteria. The detection limit was fewer than 10 bacterial cells per sample. It was also possible to detect these bacteria in dental plaque. The prevalence of these bacteria varied in each sample. The specific primers designed in this study may clarify the epidemiology of periodontal disease. [source] | |||||||||||||||||||||||||||||||||||||