POLYMERASE-CHAIN-REACTION AMPLIFICATION OF GUTHRIE CARD DEOXYRIBONUCLEIC-ACID - EXTRACTION OF NUCLEIC-ACID FROM FILTER MATRICES

This study evaluated two deoxyribonucleic acid (DNA) extraction method s for Guthrie card bloodspots. Our water-based extraction technique wa s compared with a commercial kit (GFX extraction system). In contrast to our nondenaturing method, the GFX system utilizes chaotropes to ext ract DNA. Extracted DNA is subsequently purified by selective elution from a glass fiber matrix. To evaluate DNA extraction, polymerase chai n reaction (PCR) for a. 491 bp region encoding the cystic fibrosis Del ta F508 mutation was performed and PCR products electrophoresed on pol yacrylamide gels and stained with ethidium bromide. Amplification of G FX-extracted DNA required low sample volume (1 to 5 mu L) indicating t he presence of residual PCR inhibitors. The GFX volumes (1/20 to 1/4 p unch) were comparable to our standard conditions and represented 0.16- 0.80 mu L whole blood (20 to 40 ng DNA). The GFX-extracted DNA was fou nd to be stable (6 mo, -15 degrees C). Performance time fur the GFX me thod eras less than our standard water-based extraction (about 30 min) , but more costly. The GFX extraction kit was adaptable for limited sa mple (ie, extraction of a 3 mm punch yields 20 amplifications). The GF X extraction kit provides a useful means to standardize Guthrie card D NA extraction.