A simple mouthwash method for obtaining genomic DNA in molecular epidemiological studies.
Genomic DNA for genetic analyses has traditionally been derived from blood samples. With the availability of PCR techniques requiring only minute amounts of DNA and the current demand for high-volume testing, a less invasive, simpler to perform, and cheaper method to obtain DNA is desirable. We developed a method to obtain high-quality genomic DNA from buccal cells that has high acceptability and allows for a large number of PCR assays from a single sample. Sixty subjects vigorously swished 10 ml of undiluted commercial mouthwash in the mouth for 60 s and expelled the liquid into a collection container. DNA was isolated from the buccal cells with a rapid method using proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. Electrophoretic analysis of the extracted DNA showed detectable levels of high molecular weight genomic DNA in all samples. The DNA yields ranged from 0.2 to 134.0 microg, for an average of 49.7 microg. Using these samples, all 60 subjects were successfully genotyped by PCR-based assays for polymorphisms in the CYP1A1 (MspI and exon 7), CYP2E1 (RsaI), GSTM1, GSTT1, and NQO1 genes, confirming that the quality of DNA isolated from mouthwash samples was sufficient to reliably support PCR amplification. Storage of the (unprocessed) specimens at room temperature or at 37 degrees C for 1 week (temperature conditions that may be encountered when mailing samples) or at -20 degrees C for at least 6 months did not affect the DNA yield or ability to PCR amplify the samples. The results suggest that this mouthwash procedure may be suitable for large community-based studies of genetic susceptibility to disease in which samples can be collected by the participants themselves, mailed back to the study center, and stored for months prior to DNA analysis.
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Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu 96813, USA.
Le Marchand
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D000328:Adult; D000368:Aged; D001483:Base Sequence; D004275:DNA, Ribosomal / Q000032:analysis*; D005240:Feasibility Studies; D005260:Female; D005821:Genetic Techniques; D015894:Genome, Human; D006801:Humans; D008297:Male; D008875:Middle Aged; D017720:Molecular Epidemiology / Q000379:methods; D008969:Molecular Sequence Data; D009061:Mouth Mucosa / Q000166:cytology*; D009067:Mouthwashes / Q000032:analysis; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*
D004275:DNA, Ribosomal; D009067:Mouthwashes