title: Whole genome amplification of buccal cell DNA: genotyping concordance before and after multiple displacement amplification.
abstract: While buccal cells provide an easily accessible source of genomic DNA, the amount extracted may be insufficient for many studies. Whole genome amplification (WGA) using multiple displacement amplification (MDA) may optimize buccal cell genomic DNA yield. We compared the usefulness, in epidemiological surveys, of DNA derived from buccal cells collected by alcohol mouthwash and amplified by WGA protocol and standard protocols. Buccal cell collection kits were mailed to 300 randomly selected members of a large cohort study, and 189 subjects returned buccal cell samples. We determined: (i) which QIAamp DNA Blood Mini Kit extraction protocol (tissue or blood) produced more DNA; and (ii) whether it is feasible to use MDA to prepare DNA for single nucleotide polymorphism (SNP) genotyping of markers such as the methylenetetrahydrofolate reductase (MTHFR) and vitamin D receptor (VDR) genes. The two DNA extraction protocols were tested on 20 different patient samples each. The tissue protocol yielded more DNA than the blood protocol (15.4+/-8.6 vs. 7.6+/-7.1 microg, p<0.0001). The 20 DNA samples extracted using the tissue protocol were then subjected to pre- and post-MDA genotyping using amplicons for the MTHFR SNP at C677T and the intron 8 VDR SNP. No genotyping discrepancies were detected in pair-wise comparisons of pre- and post-MDA. Genotyping DNA from MDA-based WGA is indistinguishable from routine polymerase chain reaction and offers a stable DNA source for genomic research and clinical diagnosis.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Miles D
authors.initials: MD
authors.lastname: Thompson
authors.forename: Raffick A R
authors.initials: RA
authors.lastname: Bowen
authors.forename: Betty Y L
authors.initials: BY
authors.lastname: Wong
authors.forename: Joan
authors.initials: J
authors.lastname: Antal
authors.forename: Zhanqin
authors.initials: Z
authors.lastname: Liu
authors.forename: Helen
authors.initials: H
authors.lastname: Yu
authors.forename: Katherine
authors.initials: K
authors.lastname: Siminovitch
authors.forename: Nancy
authors.initials: N
authors.lastname: Kreiger
authors.forename: Tom E
authors.initials: TE
authors.lastname: Rohan
authors.forename: David E C
authors.initials: DE
authors.lastname: Cole
mesh_terms: D001483:Base Sequence; D002610:Cheek; D015331:Cohort Studies; D004247:DNA / Q000032:analysis*; D016678:Genome; D005838:Genotype; D006801:Humans; D008297:Male; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D009061:Mouth Mucosa / Q000378:metabolism*; D009067:Mouthwashes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D018167:Receptors, Calcitriol / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D009067:Mouthwashes; D018167:Receptors, Calcitriol; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2)