Polymerase chain reaction amplification of Guthrie card deoxyribonucleic acid: extraction of nucleic acid from filter matrices.
This study evaluated two deoxyribonucleic acid (DNA) extraction methods for Guthrie card bloodspots. Our water-based extraction technique was compared with a commercial kit (GFX extraction system). In contrast to our nondenaturing method, the GFX system utilizes chaotropes to extract DNA. Extracted DNA is subsequently purified by selective elution from a glass fiber matrix. To evaluate DNA extraction, polymerase chain reaction (PCR) for a 491 bp region encoding the cystic fibrosis delta F508 mutation was performed and PCR products electrophoresed on polyacrylamide gels and stained with ethidium bromide. Amplification of GFX-extracted DNA required low sample volume (1 to 5 microL) indicating the presence of residual PCR inhibitors. The GFX volumes (1/20 to 1/4 punch) were comparable to our standard conditions and represented 0.16-0.80 microL whole blood (20 to 40 ng DNA). The GFX-extracted DNA was found to be stable (6 mo, -15 degrees C). Performance time for the GFX method was less than our standard water-based extraction (about 30 min), but more costly. The GFX extraction kit was adaptable for limited sample (i.e., extraction of a 3 mm punch yields 20 amplifications). The GFX extraction kit provides a useful means to standardize Guthrie card DNA extraction.
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
G S
GS
Makowski
E L
EL
Davis
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F
Nadeau
S M
SM
Hopfer
D001774:Blood Chemical Analysis / Q000379:methods*; D001793:Blood Preservation; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008567:Membranes, Artificial; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic
D008567:Membranes, Artificial; D011933:Reagent Kits, Diagnostic; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA