Amplification of Guthrie card DNA: effect of guanidine thiocyanate on binding of natural whole blood PCR inhibitors.
Amplification of DNA from whole blood collected on Guthrie card filter paper presents considerable technical obstacles due to the presence of natural PCR inhibitors (protein, heavy metals, heme, and heme degradation products) and low copy number of genomic material. For this purpose we evaluated guanidine thiocyanate-impregnated filter paper (GT-903), a DNA collection device designed specifically to bind PCR inhibitors and preserve DNA in an aqueous extractable form. Compared to standard 903, which retains DNA and elutes inhibitors during aqueous extraction, we found GT-903 retained 90% of protein, hemoglobin, and iron. SDS-PAGE analysis indicated that the majority of the protein released from standard 903 corresponded to albumin (70-) and globin (15-kDa); negligible levels of these proteins were eluted from GT-903. To evaluate PCR efficiency, we amplified the 491 bp region encoding the cystic fibrosis delta F508 mutation. Using comparable template, we found GT-903 amplification more efficient than standard 903 following qualitative (TBE-PAGE) and quantitative (anti-dsDNA EIA) determination. We conclude that GT-903 provides a good DNA collection device and addresses the complications associated with natural PCR inhibitors.
Makowski
G S
GS
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
Davis
E L
EL
Hopfer
S M
SM
D001665:Binding Sites / Q000187:drug effects; D001798:Blood Proteins / Q000737:chemistry / Q000187:drug effects; D001800:Blood Specimen Collection; D004247:DNA / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D005820:Genetic Testing; D006146:Guanidines / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D007501:Iron / Q000737:chemistry; D010209:Paper; D016133:Polymerase Chain Reaction / Q000379:methods*; D013861:Thiocyanates / Q000737:chemistry*
D001798:Blood Proteins; D006146:Guanidines; D006454:Hemoglobins; D013861:Thiocyanates; C054436:guanidine thiocyanate; D004247:DNA; D007501:Iron