Abstract
When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g™ WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed ≥ 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n=20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.
Original language | English (US) |
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Pages (from-to) | 303-312 |
Number of pages | 10 |
Journal | Biomarkers |
Volume | 12 |
Issue number | 3 |
DOIs | |
State | Published - May 2007 |
Externally published | Yes |
Keywords
- Genetic susceptibility
- Genotyping
- Molecular epidemiology
- Multiple displacement amplification
- TaqMan® assay
- Whole genome amplification
ASJC Scopus subject areas
- Biochemistry
- Clinical Biochemistry
- Health, Toxicology and Mutagenesis