Polymorphisms in grapevine DNA detected by the RAPD PCR technique

A sensitivie, reproducible technique is described for detecting polymorphisms between the nuclear DNA of grapevine isolates using the RAPD PCR technique. Unique fingerprints of a number of cultivars were readily distinguished by using either single primers or mixtures of two primers. The method will be used to provide a databank of fingerprints for the rapid identification of grapevine cultivars, and to develop phylogenetic relationships. It will also be extended and modified in an attempt to detect polymorphisms between DNAs of clonal selections of individual cultivars.     

Novel polyomavirus detected in the feces of a chimpanzee by nested broad-spectrum PCR

In order to screen for new polyomaviruses in samples derived from various animal species, degenerated PCR primer pairs were constructed. By using a nested PCR protocol, the sensitive detection of nine different polyomavirus genomes was demonstrated. The screening of field samples revealed the presence of a new polyomavirus, tentatively designated chimpanzee polyomavirus (ChPyV), in the feces of a juvenile chimpanzee (Pan troglodytes). Analysis of the region encoding the major capsid protein VP1 revealed a unique insertion in the EF loop of the protein and showed that ChPyV is a distinct virus related to the monkey polyomavirus B-lymphotropic polyomavirus and the human polyomavirus JC polyomavirus.     

Exon deletions and duplications in BRCA1 detected by semiquantitative PCR

rearrangements have recently been identified in the BRCA1 gene. Inclusion of a method for identifying such rearrangements should now be a prerequisite for providing a comprehensive mutation detection strategy. We have developed a semiquantitative PCR-based fluorescent assay for the detection of previously identified deletions. This method avoids the need for long PCR or Southern blotting and is suitable for large-scale epidemiological studies. The assay was used to screen 44 high-risk families within the U.K. Yorkshire Health Region. No deletions were detected, but five cases (11%) with an apparent duplication of exon 13 in BRCA1 were identified. The presence of this mutation was confirmed by long PCR. Further developments include extending the assay to include all exons of BRCA1.     

Two intragenic polymorphisms of the APC-gene detected by PCR and enzymatic digestion

Summary The gene causing adenomatous polyposis coli (APC) has recently been cloned. Three intragenic polymorphisms were reported to be detectable by single-strand conformation polymorphism analysis. Here, we describe an assay using polymerase-chain-reaction-based amplification and subsequent enzymatic digestion of genomic sequences to identify polymorphic sites at nucleotide positions 1458 and 5037 of the APC gene.     

Factor V Leiden mutation screened by PCR and detected with lanthanide-labeled probes

The Factor V Leiden mutation is an important human polymorphism, responsible for increased risk of venous thrombosis in heterozygotes as well as homozygotes. Therefore, screening is a useful possibility, and many detection systems have been described for PCR products. We have developed a simplified and robust assay using oligonucleotide probes for normal and mutant sequences, labeled with europium and samarium, respectively, and measured by time-resolved fluorescence. Populations consisting of 233 Welsh and 148 Irish subjects were examined by both restriction fragment length polymorphism (RFLP) analysis and our assay. The allele frequency was 14/466 in the Welsh and 5/296 in the Irish population, in line with other surveys of European populations. Results were not obtained in 2/381 samples by RFLP, compared with 1/381 with our method. We conclude that our method represents an improved system capable of considerable throughput at reasonable cost.     

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.     

A duplicated PLP gene causing Pelizaeus-Merzbacher disease detected by comparative multiplex PCR.

Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10%-25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP gene duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be a important genetic abnormality in PMD and affect myelin formation.     

New PCR ribotypes of Clostridium difficile detected in children in Brazil

A total of 35 Brazilian isolates of Clostridium difficile from faecal stools and four isolates from hospital environments were analyzed by PCR ribotyping. A whole cell protein profile (as an alternative for serogrouping), in vitro toxin production and susceptibility to vancomycin, metronidazole and clindamycin were also investigated. All strains were typeable by both phenotypic and genotypic methods, and a total of 13 different PCR ribotypes were identified, of which seven (132, 133, 134, 135, 136, 142 and 143) were considered new types and accounted for 78.5% of all samples evaluated (including hospital environments). A non-toxigenic C. difficile PCR ribotype 133 was detected in all children groups examined (inpatients, outpatients and healthy children), whilst toxigenic PCR ribotypes 015, 131, 134 and 135 were associated mostly with symptomatic children. Serogroups G and D were disseminated both in patients from the community and from the pediatric hospital, with group G prevalent among outpatient children. All strains were susceptible to vancomycin and metronidazole but high levels of resistance to clindamycin were found, especially among serogroups G and D. Co-existence of different ribotypes and serogroups in the same individual was observed. The new seven ribotypes found in this investigation may represent strains characteristic of this region of Brazil.     

Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends

Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal ;ladder'. However, nucleosomal ladders have only been clearly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death under physiological conditions, or by distinct molecular mechanisms. In this study, nucleosomal ladders were revealed by a ligation-mediated polymerase chain reaction (LMPCR), that amplifies DNA fragments with blunt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompany physiological cell death in mammalian tissues where previously DNA fragmentation has not been detected; (b) are produced during invertebrate cell death; (c) are invariably generated via the production of blunt, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechanisms and that the endonuclease activity is evolutionarily conserved.     

The Drosophila heat shock hsr-omega gene: an allele frequency cline detected by quantitative PCR.

The hsr-omega gene of Drosophila melanogaster produces RNA products both constitutively and at elevated levels in response to heat stress. A single-nucleotide difference in this gene that has been detected using denaturing gradient gel electrophoresis (DGGE) is responsible for an hsr-omegaa/b polymorphism, and selection experiments have indicated an association between the hsr-omegaa allele and susceptibility to heat stress. Since allele frequency estimates for population surveys using PCR and DGGE for single flies would be relatively time-consuming and expensive, we here develop a quantitative competitive-PCR method using mass-grind genomic DNA preparations for this purpose. Geographical and temporal variation of allele frequency at the hsr-omega locus in Australian populations of D. melanogaster are examined. Regular samples from a southern population through a summer season suggested stability of hsr-omegaa frequency. Field populations sampled from a approximately 2,250 km north-south transect along eastern Australia revealed a strong positive association between the frequency of hsr-omegaa and latitude, and marked spatial autocorrelation. Using appropriate analyses, strong association between population differences in hsr-omegaa frequencies and differences in temperature and rainfall measures, after controlling for latitudinal differences, support the idea that the cline in hsr-omegaa frequency may be attributable to some form of climatic selection.     

Diversity and geographical distribution of members of the Streptomyces violaceusniger 16S rRNA gene clade detected by clade-specific PCR primers

The Streptomyces violaceusniger 16S rRNA gene clade contains organisms that are of ecological interest and a rich source of novel bioactive metabolites. Improvements in the classification of members of the S. violaceusniger clade made it possible to design, evaluate and use an oligonucleotide primer set to gain an insight into the presence, distribution and taxonomic diversity of members of this taxon in environmental samples. In silico testing showed that the primers had a perfect match with representatives of the S. violaceusniger clade. The primers, designated S-S-Svio-66-a-S-20 and S-S-Svio-1274-a-A-20, amplified an approximately 1190-bp stretch of 16S rRNA gene from authenticated members of the S. violaceusniger clade, but not from representatives of other actinomycete taxa. Following amplification of DNA extracted from sediment and soil samples, the sequences of cloned PCR products confirmed the specific amplification of target sequences in 87% of the clones; the use of 16S rRNA gene fragment similarity correlations indicated that the clones represented new species. The primers can be used to facilitate the isolation of novel members of the S. violaceusniger 16S rRNA gene clade by allowing prescreening of environmental samples and the subsequent detection and retrieval of targetted strains through the use of selective isolation procedures.     

Different copy numbers of apparently identically deleted mitochondrial DNA in tissues from a patient with Kearns-Sayre syndrome detected by PCR

An apparently identical deletion of 4.977 bp in length (position 8,483-13,459) was detectable in the mitochondrial DNA from skeletal muscle, heart muscle, kidney, and liver of a patient with Kearns-Sayre syndrome. The proportion of deleted genome varied from 60% for the skeletal muscle to 15% for heart muscle and kidney, and was below 5% in the liver. The mtDNA heteroplasmy of the liver was only detectable after amplification by PCR. In skeletal and heart muscle histochemical and immunocytochemical findings concerning cytochrome c oxidase were in good correlation with the proportion of deleted mitochondrial DNA.