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PCR Products (pcr + products)
Selected AbstractsUniversal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophyELECTROPHORESIS, Issue 7 2009Chun-Chi Wang Abstract We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease. [source] Quantification of SMN1 and SMN2 genes by capillary electrophoresis for diagnosis of spinal muscular atrophyELECTROPHORESIS, Issue 13 2008Chun-Chi Wang Abstract We present the first CE method for the separation and quantification of SMN1 and SMN2 genes. Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder deleted or mutated in SMN1 gene and retained at least one copy of SMN2 gene. However, these two genes are highly homologous, differentiation and quantification of SMN1 and SMN2 are therefore required in diagnosis to identify SMA patients and carriers. We developed a fluorescence-labeled conformation-sensitive CE method to quantitatively analyze PCR products covering the variable position in the SMN1/SMN2 genes using a copolymer solution composed of hydroxyethylcellulose and hydroxypropylcellulose. The DNA samples included 24 SMA patients, 52 parents of SMA patients (obligatory carriers), and 255 controls. Those 331 samples were blind analyzed to evaluate the method, and the results compared with those obtained using denaturing HPLC (DHPLC). Validation of accuracy was performed by comparing the results with those of DHPLC. Nine of total samples showed different results. Diagnosis of one fetus DNA among them was related to abortion or not, which was further confirmed by gel electrophoresis and DNA sequencing. Our method showed good coincidence with them, and proved the misdiagnosis of DHPLC. This simple and reliable CE method is a powerful tool for clinical genotyping of large populations to detect carriers and SMA patients. [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] Semiquantitative determination of Alicyclobacillus acidoterrestris in orange juice by reverse- transcriptase polymerase chain reaction and capillary electrophoresis , laser induced fluorescence using microchip technologyELECTROPHORESIS, Issue 21-22 2004Maribel Funes-Huacca Abstract The semiquantitative detection of Alicyclobacillus acidoterrrestris in orange juice by reverse-transcriptase polymerase chain reaction (RT-PCR) with a linear dynamic range of 2×105, 2 colony forming units (CFU)/mL in terms of cell count is described. Separation, detection, and quantification of the RT-PCR products were accomplished using the Agilent 2100 bioanalyzer in conjunction with the DNA 1000 LabChip kit. After 0 and 12 h of enrichment, it was possible to generate a linear standard curve between the amount of cells and amplicon concentration of RT-PCR and PCR products. Using this method, cell diminution was verified in samples of orange juice treated with a natural inhibitor (Sapindus saponaria), determining the persistence of viable cells. Semiquantitative RT-PCR using the Agilent 2100 bioanalyzer is a potentially useful approach for rapid in vitro determination of A. acidoterrestris and monitoring of inhibitor susceptibility for the orange juice-producing industry. [source] Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescenceELECTROPHORESIS, Issue 14 2004Virginia García-Cañas Abstract The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5,0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge, these results demonstrate for the first time that multiplex PCR-CGE-LIF is a solid alternative to determine multiple genetically modified organisms in maize flours in a single run. [source] Dynamics of marine bacterial and phytoplankton populations using multiplex liquid bead array technologyENVIRONMENTAL MICROBIOLOGY, Issue 4 2010Xavier Mayali Summary Heterotrophic bacteria and phytoplankton dominate the biomass and play major roles in the biogeochemical cycles of the surface ocean. Here, we designed and tested a fast, high-throughput and multiplexed hybridization-based assay to detect populations of marine heterotrophic bacteria and phytoplankton based on their small subunit ribosomal DNA sequences. The assay is based on established liquid bead array technology, an approach that is gaining acceptance in biomedical research but remains underutilized in ecology. End-labelled PCR products are hybridized to taxon-specific oligonucleotide probes attached to fluorescently coded beads followed by flow cytometric detection. We used ribosomal DNA environmental clone libraries (a total of 450 clones) and cultured isolates to design and test 26 bacterial and 10 eukaryotic probes specific to various ribotypes and genera of heterotrophic bacteria and eukaryotic phytoplankton. Pure environmental clones or cultures were used as controls and demonstrated specificity of the probes to their target taxa. The quantitative nature of the assay was demonstrated by a significant relationship between the number of target molecules and fluorescence signal. Clone library sequencing and bead array fluorescence from the same sample provided consistent results. We then applied the assay to a 37-day time series of coastal surface seawater samples from the Southern California Bight to examine the temporal dynamics of microbial communities on the scale of days to weeks. As expected, several bacterial phylotypes were positively correlated with total bacterial abundances and chlorophyll a concentrations, but others were negatively correlated. Bacterial taxa belonging to the same broad taxonomic groups did not necessarily correlate with one another, confirming recent results suggesting that inferring ecological role from broad taxonomic identity may not always be accurate. [source] Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex methodENVIRONMENTAL MICROBIOLOGY, Issue 10 2008David M. Gordon Summary It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85,90% of E. coli strains can be assigned to a phylo-group and that 80,85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group. [source] Analysis of the distribution and diversity in recent Hawaiian volcanic deposits of a putative carbon monoxide dehydrogenase large subunit geneENVIRONMENTAL MICROBIOLOGY, Issue 9 2005Kari E. Dunfield Summary A putative carbon monoxide dehydrogenase large subunit gene (BMS putative coxL) was amplified from genomic DNA extracts of four recent (42,300 year old) Hawaiian volcanic deposits by polymerase chain reaction (PCR). Sequence databases derived from clone libraries constructed using PCR products were analysed phylogenetically and statistically. These analyses indicated that each of the deposits supported distinct BMS putative coxL gene assemblages. Statistical analyses also showed that the youngest deposit (42 years old) contained the least diverse sequences (P < 0.05), but that diversity did not vary significantly among three older deposits with ages from about 108,300 years. Although diversity indices did not vary among the older deposits, mismatch analyses suggested population structures increased in complexity with increasing deposit age. At each of the sites, most of the clone sequences appeared to originate from Proteobacteria not currently represented in culture or recognized as CO oxidizers. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysisENVIRONMENTAL TOXICOLOGY, Issue 6 2001Judith A. Baker Abstract We report molecular analyses which identify cyanobacterial strains present in environmental samples. These analyses do not require the isolation and culture of strains. Identification of cyanobacteria used the polymerase chain reaction (PCR), based on the phycocyanin operon. Differentiation was either by restriction endonuclease digestion (restriction fragment length polymorphisms) or sequencing of the PCR products. Identification was based on sequence homology of the intergenic spacer region (IGS) between the ,- and ,-phycocyanin subunits (PC-IGS) with database records. We have found that the length and sequence of the PC-IGS is capable of predicting the genus accurately, but not the species. Toxigenicity was determined with oligonucleotide probes for key steps in the microcystin toxin synthesis pathway. We have shown that it is possible to easily and routinely obtain PCR amplification products and differentiate the strains in bloom samples. The methods can detect even minor components in bloom samples, which may not be apparent on microscopic examination. Genetic probes for microcystin toxigenicity are effective on environmental samples, eliminating the need for isolation and culture of the organisms. The use of a suite of tests described here will allow water managers to determine the presence and the type of cyanobacteria and their microcystin toxigenicity. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 472,482, 2001 [source] Human alcoholism studies of genes identified through mouse quantitative trait locus analysisADDICTION BIOLOGY, Issue 4 2002Marissa A. Ehringer Coding region DNA sequence variants have been recently identified in several QTL candidate genes in a mouse model of differential sensitivity to alcohol [inbred long-sleep (ILS) and inbred short-sleep (ISS)]. This work has been extended into a human population characterized for their initial level of response to alcohol (LR). The coding region of one of the most promising of these candidate genes, zinc finger 133 (Znf133), has been sequenced completely in 50 individuals who participated in alcohol challenges at approximately age 20 and have been followed subsequently for the last 15 years. PCR products were obtained for the protein coding region of ZNF133 using human genomic DNA and directly sequenced using automated sequencers. Novel single nucleotide polymorphisms (SNPs) were detected by analyzing the sequence data using a suite of bioinformatics programs including Consed, Phred, Phrap and Polyphred. Five human SNPs were detected, two that correspond to amino acid changes in the protein, two that are silent DNA changes and one located in an intron. In this small sample, no significant association between any of the SNPs and alcohol diagnosis was detected. A follow-up of these SNPs in a larger sample should allow a more definitive conclusion to be reached. Significantly, the data presented here demonstrate the feasibility of directly testing genes in human alcoholic populations that had been identified first by comparative DNA sequencing of candidate genes located within mouse alcohol-related QTLs, even without detailed knowledge of the gene's function. [source] Novel non-sense GCH1 mutation in a South African family diagnosed with dopa-responsive dystoniaEUROPEAN JOURNAL OF NEUROLOGY, Issue 3 2010S. Bardien Background:, Dopa-responsive dystonia (DRD), a movement disorder characterized by onset in early childhood and a dramatic response to low doses of levodopa, has been shown to be caused by a number of different mutations in the GCH1 gene. Methods:, We identified a South African family which presented with DRD in three family members. Polymerase chain reaction (PCR) primers were designed to span all six exons of GCH1 and the PCR products were screened for pathogenic mutations using direct sequencing. Results:, A novel non-sense mutation (c.233delT; p.I78fsX79) was identified in the DRD patients, which would produce a markedly truncated protein of only 78 amino acids. This mutation was also present in a number of asymptomatic family members. Conclusions:, A novel non-sense mutation in the GCH1 gene can be associated with DRD and reduced penetrance in South African patients. [source] Multilineage progression of genetically unstable tumor subclones in cutaneous T-cell lymphomaEXPERIMENTAL DERMATOLOGY, Issue 8 2004Albert Rübben Abstract:, Molecular analysis of solid malignant tumors has suggested multilineage progression of genetically unstable subclones during early stages of tumorigenesis as a common mechanism of tumor cell evolution. We have investigated whether multilineage progression is a feature of cutaneous T-cell lymphoma (CTCL). To identify individual tumor cell subclones, we determined the pattern of mutations within microsatellite DNA obtained from multiple histomorphologically confined tumor cell nests of mycosis fungoides (MF) and lymphomatoid papulosis (LyP) lesions. Tumor cells were isolated by laser microdissection, and allelotypes were determined at microsatellite markers D6S260, D9S162, D9S171, D10S215, TP53.PCR15, and D18S65. Nine cases of MF and one patient with anaplastic large cell lymphoma (ALCL) originating from LyP were analyzed at 277 different microdissected areas obtained from 31 individual lesions. Three specimens of cutaneous lichen planus microdissected at 26 areas served as the control tissue. Microsatellite instability in microdissected tissue [MSI(md-tissue)] was detected in tumor tissues of all CTCL patients. One hundred and fifty-seven of 469 analyzed polymerase chain reaction (PCR) amplifications contained mutated microsatellite alleles (34%). In lichen planus, MSI(md-tissue) was seen in only four of 76 PCR products (5%) (P < 0.0001). The distribution of allelotypes in tumor cells from different disease stages was consistent with multilineage progression in five MF cases, as well as in the LyP/ALCL patient. Our results suggest that CTCL may evolve by multilineage progression and that tumor subclones in MF can be detected in early disease stages by mutation analysis of microsatellite DNA obtained from multiple microdissected areas. [source] Genomic organization and amplification of the human plakoglobin gene (JUP)EXPERIMENTAL DERMATOLOGY, Issue 5 2000N. V. Whittock Abstract: Plakoglobin is a globular protein common to the intracellular plaques of adhesive junctions, predominantly desmosomes and adherens junctions. Recently, a number of pathogenic mutations have been described in other components of desmosomes, specifically in plakophilin 1, desmoplakin and desmoglein 1. The phenotype of affected patients mainly involves thickening of palm and sole skin (keratoderma). Although no human mutations in plakoglobin have been described thus far, this protein represents an excellent candidate for other human genetic disorders, possibly involving skin and heart, sites of high plakoglobin expression. To facilitate future mutation detection analyses in such conditions, we have characterized the intron,exon organization of the human plakoglobin gene, which comprises 13 distinct exons spanning approximately 17 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products. [source] agr -Genotyping and transcriptional analysis of biofilm-producing Staphylococcus aureusFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2007Viviana Cafiso Abstract We investigated the correlation between biofilm production and the accessory-gene-regulator (agr) in 29 strains isolated from catheter-associated infections compared to a control group (30 isolates). All strains were tested for their ability to produce biofilm in a static system, and their agr genotype was determined. ScaI-restriction fragment length polymorphism for agr -typing showed that strong biofilm-producing strains belong to agr - type II. We found two new agr -variants, and sequence analysis of the three PCR products revealed the insertion of IS256 within the agr- locus. Biofilm production was assessed and correlated with agr functionality, with the expression of the ica -operon and of two transcriptional regulators, sarA and rsbU. Our data show that agr -II strains produce large amounts of biofilm, possess a defective agr -system show early transcription of icaA and are defective in haemolysin activity, icaR transcription, and in the expression of the ,B activator rsbU. Strains with agrIII are medium biofilm producers, have an inactive agr -system, but express icaAR and rsbU in the late- and postexponential growth phases. In agrI,IV- and -IA-variants, medium or weak biofilm production was found. In these strains, the agr -locus was fully functional, rsbU- icaR and icaA were found in the late- and/or postexponential phases. Biofilm production was not affected by sarA. [source] Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter speciesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2005Waleed Abu Al-Soud Abstract The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 °C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile. [source] cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groupsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2005Mohamed Ramelah Abstract Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3, region of cagA gene were examined among 192 Malaysian H. pylori cagA -positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621,651 bp), type B (732,735 bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p > 0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific- cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p < 0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p < 0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection. [source] New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatusFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2001Camile Pizeta Semighini Abstract In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical. [source] Microbial community dynamics in nutrient-pulsed chemostatsFEMS MICROBIOLOGY ECOLOGY, Issue 1 2006Militza Carrero-Colón Abstract In nature, microbes are subject to nutrient fluxes. As the periodicity of nutrient flux lengthens, different physiological traits may be selected. The competitive exclusion principle stipulates that one organism will dominate these systems; however, interspecies interactions may produce a dynamic microbial community. These issues were investigated in chemostats pulsed with gelatin. Chemostats were run over 30 days with substrate addition continuously or at intervals of 0.5, 1 or 3 days. Growth rates were similar between pulse intervals. Ectoaminopeptidase activity levels remained relatively constant within a pulse interval. Bacterial community structure was monitored using denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene. There were dynamic changes at all periodicities; however, the pace of these changes decreased over time. Final communities were not identical between different treatments. The structure of persistent vs. active microbial populations was compared by denaturing gradient gel electrophoresis of the PCR and reverse transcriptase-PCR amplicons of 16S rDNA and rRNA templates, respectively. For all the chemostats, the rRNA profiles were not identical to the rDNA profiles for a sample. These experiments demonstrate that complex community dynamics can occur under environmental heterogeneities that are modest relative to those found in natural aquatic habitats. Furthermore, the physiological functionality of these dynamic communities was stable. [source] Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolatesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2005Sarita Sarita Abstract A group-specific primer set was developed using nodC as a target gene for the amplification of rhizobial sequence diversity from nodule isolates and total soil DNA preparations. The primer set was tested on 209 nodule isolates, recovered from six different trap plant species which were grown in two soil samples collected from a chickpea and a wheat field site in India. We also amplified and cloned PCR products from total DNA isolated from the same soil samples. The total diversity within the resulting clone libraries (, 218 clones) was higher than that recovered from trap plants, but differed depending on the PCR protocols and primers used. However, some plant-selected genotypes could not be obtained using the community approach, probably due to variable detection limits and limited clone library sizes. [source] Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoideaFEMS YEAST RESEARCH, Issue 5 2008Mette D. Jacobsen Abstract We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I. [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] 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] Microbial diversity of a sulphide spire located in the Edmond deep-sea hydrothermal vent field on the Central Indian RidgeGEOBIOLOGY, Issue 2 2003Joost Hoek ABSTRACT A culture-independent molecular phylogenetic survey was carried out for a bacterial and archaeal community of a mineralized crust coating a sulphide spire, which was collected from the Edmond vent field (23° S, 69° E, 3300 m depth) on the Central Indian Ridge. Small-subunit rRNA genes (16S rDNA) were amplified from environmental DNA by PCR utilizing Bacteria-specific, and Archaea-specific 16S rDNA primers. PCR products were cloned and 26 bacterial and nine archaeal unique sequence types (phylotypes) were identified from 150 clones analysed by restriction fragment length polymorphism, representing eight and four distinct lineages, respectively. The majority (>90%) of the bacterial phylotypes group with the ,-Proteobacteria and confirms the global prevalence of ,-Proteobacteria in deep-sea hydrothermal environments. Among the ,-Proteobacteria, >40% of the phylotypes were closely related to the recently isolated deep-sea vent thermophilic chemolithoautotrophic sulphur-reducer, Nautilia lithotrophica. A single bacterial sequence was nearly identical (99% similarity) to the thermophilic hydrogen-oxidizing Hydrogenobacter thermolithotrophum, and is the first report of Hydrogenobacter at deep-sea hydrothermal vents. A majority (97%) of the archaeal phylotypes grouped with the ,Deep-sea Hydrothermal Vent Euryarchaeotal Group', a phylogenetic lineage of uncultured Archaea that have only been reported from other deep-sea hydrothermal vents on the Mid-Atlantic Ridge, East Pacific Rise, Juan de Fuca Ridge, Isu,Ogasawara Arc, Okinawa Trough and the Manus Basin. A single sequence was closely related to the hyperthermophilic sulphur-reducing Thermococcales frequently found in diverse deep-sea vent environments. Scanning electron micrographs of the mineralized crust reveal abundant filamentous, rod and coccoidal forms encased in sulphur and sulphide mineral precipitate, suggesting that the thermophilic chemolithoautorophs and sulphide-producing heterotrophs may influence the architecture and sulphur cycling of the sulphide spire. [source] Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigreeHAEMOPHILIA, Issue 6 2009T. YU Summary., Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572-1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband's F7 gene. To investigate the splicing patterns associated with F7 c.572-1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position -1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT-PCR analysis. The aberrant transcripts from the F7 c.572-1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT-PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572-1G>A mutant allele is responsible for the factor VII deficiency in this pedigree. [source] Prevalence of Duodenal Ulcer-Promoting Gene (dupA) of Helicobacter pylori in Patients with Duodenal Ulcer in North Indian PopulationHELICOBACTER, Issue 6 2007H. S. Jayasinghe Arachchi Abstract Background: , The duodenal ulcer (DU)-promoting gene (dupA) of Helicobacter pylori has been identified as a novel virulent marker associated with an increased risk for DU. The presence or absence of dupA gene of H. pylori present in patients with DU and functional dyspepsia in North Indian population was studied by polymerase chain reaction (PCR) and hybridization analysis. Materials and Methods: , One hundred and sixty-six patients (96 DU and 70 functional dyspepsia) were included in this study. In addition, sequence diversity of dupA gene of H. pylori found in these patients was analyzed by sequencing the PCR products jhp0917 and jhp0918 on both strands with appropriate primers. Results: , PCR and hybridization analyses indicated that dupA gene was present in 37.5% (36/96) of H. pylori strains isolated from DU patients and 22.86% (16/70) of functional dyspepsia patients (p .05). Of these, 35 patients with DU (97.2%) and 14 patients with functional dyspepsia (81.25%) were infected by H. pylori positive for cagA genotype. Furthermore, the presence of dupA was significantly associated with the cagA -positive genotype (p .02). Conclusion: , Results of our study have shown that significant association of dupA gene with DU in this population. The dupA gene can be considered as a novel virulent marker for DU in this population. [source] Non- pylori Helicobacteraceae in the Upper Digestive Tract of Asymptomatic Venezuelan Subjects: Detection of Helicobacter cetorum- like and Candidatus Wolinella africanus -like DNAHELICOBACTER, Issue 5 2007M. Alexandra García-Amado Abstract Background: The spectrum of human non- pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non- pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. Materials and methods: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter- like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18,68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. Results: We detected DNA sharing 99,100% sequence identity in over 300,400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). Conclusions: Our results suggest that non- pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus. [source] New Approaches for Validation of Lethal Phenotypes and Genetic Reversion in Helicobacter pyloriHELICOBACTER, Issue 1 2001Timothy K. McDaniel Background. Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Materials and Methods. We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. Results. After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. Conclusions. The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori. [source] Cocirculation of and coinfections with hepatitis A virus subgenotypes IIIA and IB in patients from Pune, western IndiaHEPATOLOGY RESEARCH, Issue 2 2007Shobha Chitambar Aim:, During the 1990s, a changing pattern of epidemiology of hepatitis A was reported in different populations of India. The present study was undertaken to investigate the molecular epidemiology of hepatitis A virus (HAV) strains over a period of 10 years. Methods:, Stool/serum samples were collected from hepatitis A patients clinically presenting acute viral hepatitis and hepatic encephalopathy. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect HAV-RNA. HAV genomes were examined by sequencing PCR products of VP1/2A junction (168 bp) and RNA polymerase (116 bp) regions. Results:, Subgenotype IIIA and IB were detected in 74.2% and 9.7% of specimens, respectively, while 16.1% of patients had mixed infections. Sewage samples also showed presence of both IIIA (9/10) and IB (1/10) subgenotypes. RNA polymerase region showed two clusters constituting 51.6% and 19.4% strains closer to Nor21 and HM175 strains, respectively, in clinical specimens. Three isolates appeared as discordant subgenotypes in VP1/2A and RNA polymerase regions. Conclusion:, The data revealed cocirculation of and coinfection with subgenotypes IIIA and IB, with predominance of IIIA and genetic heterogeneity of HAV strains in western India. [source] Systematic evaluation of the effect of common SNPs on pre-mRNA splicing,HUMAN MUTATION, Issue 4 2009Abdou ElSharawy Abstract The evolutionary and biomedical importance of differential mRNA splicing is well established. Numerous studies have assessed patterns of differential splicing in different genes and correlated these patterns to the genotypes for adjacent single-nucleotide polymorphisms (SNPs). Here, we have chosen a reverse approach and screened dbSNP for common SNPs at either canonical splice sites or exonic splice enhancers (ESEs) that would be classified as putatively splicing-relevant by bioinformatic tools. The 223 candidate SNPs retrieved from dbSNP were experimentally tested using a previously established panel of 92 matching DNAs and cDNAs. For each SNP, 16 cDNAs providing a balanced representation of the genotypes at the respective SNP were investigated by nested RT-PCR and subsequent sequencing. Putative allele-dependent splicing was verified by the cloning of PCR products. The positive predictive value of the bioinformatics tools turned out to be low, ranging from 0% for ESEfinder to 9% (in the case of acceptor-site SNPs) for a recently reported neural network. The results highlight the need for a better understanding of the sequence characteristics of functional splice-sites to improve our ability to predict in silico the splicing relevance of empirically observed DNA sequence variants. Hum Mutat 0, 1,9, 2009. © 2009 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||