Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies

Biomarkers. 2007 May-Jun;12(3):303-12. doi: 10.1080/13547500601162011.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Adult
  • Aged
  • Algorithms
  • DNA / analysis*
  • DNA / genetics
  • DNA / isolation & purification
  • Female
  • Genome, Human*
  • Genotype
  • Humans
  • Leukocytes / metabolism
  • Male
  • Middle Aged
  • Molecular Epidemiology
  • Mouth Mucosa / metabolism*
  • Nucleic Acid Amplification Techniques / methods*
  • Polymorphism, Single Nucleotide

Substances

  • DNA