Alex Renwick | PubMed Health

PubMed2005

Microbial DNA typing by automated repetitive-sequence-based PCR.

Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.

Automation↗

PubMed2002

Complex SNP-based haplotypes in three human helicases: implications for cancer association studies.

We have initiated a candidate gene approach to study variation and predisposition to cancer in the four major ethnic groups that constitute the U.S. population (African Americans, Caucasians, Hispanics, and Asians). We resequenced portions of three helicase genes (BLM, WRN, and RECQL) identifying a total of 37 noncoding single nucleotide polymorphisms (SNPs). Haplotype inference predicted 50 haplotypes in BLM, 56 in WRN, and 47 in RECQL in a sample of 600 chromosomes. Approximately 10% of the predicted haplotypes were shared among all ethnic groups. Linkage disequilibrium and recombination effects showed that each locus has taken a diverse evolutionary path. Primate DNA analysis of the same loci revealed one human haplotype per gene shared with the great apes, indicating that the observed diversity occurred since the divergence of humans from the last common ancestor. In BLM, we confirmed the presence of a founder haplotype among Ashkenazi Jews homozygous for the blm(Ash) mutation. The cosegregating haplotype was seen in all (6/6) samples of Ashkenazi descent, whereas in the general population it has a low frequency (0.02) and was not found in African Americans. In WRN, ethnic samples were studied for their haplotype content and the presence or absence of six previously described coding SNPs (cSNPs). Hispanic individuals carrying two of these cSNPs showed a 60% increase in the frequency of a common haplotype (haplotype No. 28). In the pooled sample, no association was found. Because (1) the majority of the haplotypes are population specific and (2) the patterns of linkage disequilibrium, recombination, and haplotype diversity are markedly different between gene regions, these data show the importance of either ethnically matched controls or within-family-based disease-gene association studies.

Alleles↗