Long-term storage and recovery of buccal cell DNA from treated cards.
Economical methods for collecting and storing high-quality DNA are needed for large population-based molecular epidemiology studies. Buccal cell DNA collected via saliva and stored on treated filter paper cards could be an attractive method, but modest DNA yields and the potential for reduced recovery of DNA over time were unresolved impediments. Consequently, buccal cell DNA collection via oral mouthwash rinsing became the method of choice in epidemiologic studies. However, the amount of genomic DNA (gDNA) required for genotyping continues to decrease, and reliable whole genome amplification (WGA) methods further reduced the mass of gDNA needed for WGA to 10 ng, diminishing the obstacle of low DNA yields from cards. However, concerns about yield and DNA quality over time remained. We located and analyzed 42 buccal cell saliva samples collected and stored on treated cards for 7 years at room temperature, -20 degrees C, and -80 degrees C. We recovered DNA from the treated cards, estimated the concentration by a human-specific quantitative real-time PCR assay, and evaluated the quality by PCR amplification of 268-, 536-, and 989-bp fragments of the beta-globin gene and by AmpFlSTR Identifiler assay analysis. Most DNA yields per 3-mm punch were <10 ng, and most PCR amplicons failed to amplify, where size of the amplicon was negatively associated with successful amplification. Using these methods, treated cards did not consistently provide sufficient quantities of buccal cell gDNA after 7 years of storage for genotyping or WGA.
Sigurdson
Alice J
AJ
Radiation Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, 6120 Executive Boulevard, EPS 7092, MSC 7238, Bethesda, Maryland 20892-7238, USA. sigurdsa@mail.nih.gov
Ha
Mina
M
Cosentino
Mark
M
Franklin
Tracie
T
Haque
Kashif A
KA
Qi
Ying
Y
Glaser
Cynthia
C
Reid
Yvonne
Y
Vaught
Jim B
JB
Bergen
Andrew W
AW
D004247:DNA / Q000032:analysis*; D006801:Humans; D009061:Mouth Mucosa / Q000166:cytology*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D013048:Specimen Handling / Q000191:economics / Q000295:instrumentation / Q000379:methods*
D004247:DNA