Sequence-based Polymerase Chain Reaction Research Articles

Childhood-onset primary open angle glaucoma in a Canadian kindred: clinical and molecular genetic features

Objective: To describe the clinical features and identify the molecular etiology of childhood-onset primary open angle glaucoma (POAG) in a Canadian kindred. Methods: Members of a Canadian Caucasian family with POAG were examined and DNA obtained. Single-strand conformation polymorphism analysis was performed using reported primers from exon 3 of the myocilin gene. A single-stranded conformation polymorphism was characterized by polymerase chain reaction-based sequencing. Results: Two affected half-sibs had onset of severe glaucoma at age 3 and 9. Their mother had lost vision in one eye from glaucoma by age 17. All three affected subjects had undergone bilateral glaucoma filtering surgery. Both fathers were unaffected. A single-stranded conformation polymorphism was identified in the mother and the two affected daughters and was absent in one father. A single base change from C?T at nucleotide position 1109 was identified in the affected members of the family by direct sequencing. This mutation, which causes a nonconservative amino acid change (Pro370Leu), was not found on 192 normal chromosomes from Caucasian individuals. Conclusion: We report a Canadian family with childhood-onset, severe POAG due to a mutation in the myocilin gene.

Repetitive element sequence based polymerase chain reaction for typing of Brucella strains

A collection of 40 Brucella strains, including 19 clinical B.canis isolates, was analyzed using ERIC-PCR and REP-PCR. PCR amplification using primers REP 1R-I and REP 2-I provided distinct patterns for Brucella spp. All Brucella strains could be discriminated at least to the species level. ERIC-PCR, employing ERIC 1R and ERIC 2 as primers was less discriminative for Brucella, only allowing a discrimination to the genus level with occasional discrimination between individual strains. It can be concluded that rep PCR is a promising fingerprinting method for the evaluation of an outbreak.

Molecular diversity of the HLA-C locus in unrelated marrow transplantation.

The diversity of HLA-C exon-2 alleles in 56 HLA-A, B, DRB and DQB1-matched patient-unrelated marrow donor pairs was examined by non-cloning polymerase chain reaction-based sequencing of genomic DNA. This method allows simultaneous analysis of both alleles in heterozygous samples. All Cw5-positive individuals encoded a sequence which differed from the published Cw*0501 sequence at position 61. Among 82 samples assigned a single antigen by serologic testing, 64 (78%) were heterozygous for two distinct alleles when tested by sequencing. Cw*1202, 1601 and 15 were identified in samples for which no phenotype could be assigned (C "blank"). Finally, 7 of the 56 HLA-A, B, DRB, DQB1-matched pairs (12.5%) were mismatched for one or both HLA-C alleles. We conclude that sequence-based methods constitute the optimal strategies for typing HLA-C alleles in the unrelated marrow transplant population.

Presymptomatic diagnosis of familial adenomatous polyposis coli

Familial adenomatous polyposis (FAP), an autosomal dominant inherited disease, confers a high risk of colon cancer, and recently the gene responsible for FAP, termed adenomatous polyposis coli (APC) gene, was identified and fully characterized. For the presymptomatic diagnosis of FAP, we have performed linkage studies using two polymorphic systems close to or at the APC locus; cytosine-adenine dinucleotide repeat length polymorphism and restriction endonuclease RsaI site polymorphism. Based on the two polymorphic systems, we have determined the haplotype at the APC locus in 23 individuals of two Korean families with FAP. From these haplotypes of individuals, we could make the diagnosis, whether affected or unaffected, in 74 percent of 31 at-risk persons. To decrease the chance of misdiagnosis caused by recombinant events, the use of haplotypes was better than using one polymorphic system. In addition to polymorphic analysis, we have also searched germline mutations of the APC gene in eight individuals (26 percent of all 31 at risk persons) of another two FAP families which could not be diagnosed definitely by linkage analysis. A 5 base-pairs deletion at codon 1309 was detected in one of the families, and a 5 base-pairs deletion at codon 1185 was also identified in another family by using a ribonuclease protection assay followed by DNA sequencing. From these results, we could diagnose FAP with 100 percent accuracy. Linkage studies by the RsaI site polymorphism and cytosine-adenine repeat length polymorphism as well as the polymerase chain reaction-based sequencing method provide accurate and efficient tools for presymptomatic diagnosis of FAP in their families.

Molecular Epidemiology of a Parainfluenza Type 3 Virus Outbreak on a Pediatric Ward

Parainfluenza type 3 virus (PIV-3), an important cause of acute lower respiratory illness in children, can be transmitted nosocomially. To differentiate between nosocomial transmission and community-acquired infection, a polymerase chain reaction-based sequencing assay was developed for the 5' noncoding region of the PIV-3 fusion protein gene and was applied to virus specimens from 10 children infected with PIV-3 during a hospital outbreak. Four strains of PIV-3 were identified among the 10 virus isolates. Six isolates, which appeared to belong to 1 strain, were obtained from a cluster of nosocomial cases in a pediatric intermediate care unit. In contrast, the remaining 4 isolates, which appeared to belong to 3 different strains, were obtained from children infected in the community or elsewhere in the hospital. These data indicate that multiple strains of PIV-3 can be found during a single epidemic and provide evidence that infections within the intermediate care unit were probably caused by transmission of 1 strain of virus within the unit rather than reintroduction of virus by new patients or staff.

19] Transposon-based and polymerase chain reaction-based sequencing of DNAs cloned in a phage

Transposon- and polymerase chain reaction (PCR)-based methods for sequencing DNAs cloned in λ phage vectors hold great promise for many DNA sequencing projects. Three sets of steps are involved, each of which is automatable: (1) transposon insertion, (2) direct and crossover PCR from transposon containing phage to map insertions and generate sequencing templates, and (3) linear amplification sequencing of appropriate PCR fragments. This strategy yields accurate high-resolution sequence ladders and is cost- and labor-efficient. More development is needed, however, to realize the full potential of this transposon- and PCR-based approach. The two most serious current limitations are an undersupply of Tn5 sup F insertions in certain target regions and an inability to reproducibly PCR amplify over long enough distances to map all insertions of interest in λ clones in one attempt. Each of these limitations will probably be overcome in the near future.

Phylogeny of the phototrophic rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment

A 260-bp segment of the DNA that encodes 16S rRNA, corresponding to positions 44 to 337 in the Escherichia coli sequence, was amplified by the polymerase chain reaction and sequenced from each of 13 bacteria (rhizobia and purple phototrophs) in the alpha subdivision of the class Proteobacteria. The phylogenetic tree calculated from differences in the sequenced segment conforms well to our expectations based on other previously published data. The sequence from BTAi1 (a recently described phototrophic symbiont of the legume Aeschynomene) and that from the free-living phototroph Rhodopseudomonas palustris both fall within the range of variation found among strains of the soybean symbiont Bradyrhizobium japonicum. This suggests that it would be appropriate to include all of these organisms in a single genus.

Differentiation of Mycobacterium species by direct sequencing of amplified DNA

Nucleotide sequences specific for a range of Mycobacterium species were defined by computer-assisted sequence comparisons of small subunit ribosomal RNA. A polymerase chain reaction-based sequencing strategy was used to demonstrate that the 16S rRNA sequence can be used for the rapid identification of mycobacterial isolates. Identification at the species level can be obtained within 2 d, requiring less than 10,000 bacteria. This procedure reliably differentiates Mycobacterium spp. which are difficult to identify by classical methods, such as M. malmoense, M. szulgai and M. flavescens.