Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.
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.
Moore
L E
LE
Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20852-7240, USA. moorele@mail.nih.gov
Bergen
A W
AW
Haque
K A
KA
Qi
Y
Y
Castle
P
P
Chanock
S J
SJ
Egan
K
K
Newcomb
P
P
Titus-Ernstoff
L
L
Alguacil
J
J
Rothman
N
N
Garcia-Closas
M
M
D000328:Adult; D000368:Aged; D000465:Algorithms; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D017720:Molecular Epidemiology; D009061:Mouth Mucosa / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D004247:DNA