Serum, plasma and paraffin-embedded tissues as sources of DNA for studying cancer susceptibility genes.
The ability to isolate DNA from archived human serum, plasma and paraffin-embedded human tissues enhances opportunities to study breast, lung and other cancer risk factors. We report herein a simple and fast protocol for the extraction of genomic DNA from these sources. Using a phenol-based extraction method, the recovery for DNA is quantitative and reproducible. DNA yields in serum (250 microl) were between 162 and 1060 ng (n = 18 subjects), in plasma (250 microl) were between 165 and 375 ng (n = 5 subjects) and in embedded tissues (5-microm thick sections for ethanol fixed, and between 5- and 20-microm sections for formaldehyde fixation) were between 1 microg and 11.7 microg (n = 32 subjects). The extraction method was combined with newly designed PCR-based assays for cancer susceptibility marker genes such as CYP1A1 (exon 7), CYP2E1 (Dra1, Rsa1), GSTM1 and NAT2 [NAT2*5A (C481T), NAT2*6A (G590A), NAT2*7A (G857A)]. Genotyping results from the serum and paraffin-embedded tissues compared favorably to results from archived freshly frozen tissues, where concordance was 98% for serum, 100% for ethanol-fixed embedded tissues, and 97% for formaldehyde-fixed and paraffin-embedded tissues. This facile method will allow for the use of archived tissue samples of prospective cohort and other studies where intact DNA was not previously available.
Blömeke
B
B
Laboratory of Human Carcinogenesis, National Cancer Institute, NIH Bethesda, MD 20892, USA.
Bennett
W P
WP
Harris
C C
CC
Shields
P G
PG
D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D002273:Carcinogens / Q000493:pharmacokinetics; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000097:blood / Q000302:isolation & purification*; D004273:DNA, Neoplasm / Q000097:blood / Q000302:isolation & purification; D005091:Exons; D020022:Genetic Predisposition to Disease; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008658:Inactivation, Metabolic; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000201:enzymology / Q000502:physiology; D008175:Lung Neoplasms / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D009857:Oncogenes; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results
D002273:Carcinogens; D004273:DNA, Neoplasm; D007527:Isoenzymes; D004247:DNA; D019392:Cytochrome P-450 CYP2E1; D019363:Cytochrome P-450 CYP1A1; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human; D005982:Glutathione Transferase