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 o f natural PCR inhibitors (protein, heavy metals, heme, and heme degrad ation products) and low copy number of genomic material. For this purp ose we evaluated guanidine thiocyanate-impregnated filter paper (GT-90 3), a DNA collection device designed specifically to bind PCR inhibito rs and preserve DNA in an aqueous extractable form. Compared to standa rd 903, which retains DNA and elutes inhibitors during aqueous extract ion, we found GT-903 retained 90% of protein, hemoglobin, and iron. SD S-PAGE analysis indicated that the majority of the protein released fr om standard 903 corresponded to albumin (70-) and globin (15-kDa); neg ligible levels of these proteins were eluted from GT-903. To evaluate PCR efficiency, we amplified the 491 bp region encoding the cystic fib rosis Delta F508 mutation. Using comparable template, we found GT-903 amplification more efficient than standard 903 following qualitative ( TEE-PAGE) and quantitative (anti-dsDNA EIA) determination. We conclude that GT-903 provides a good DNA collection device and addresses the c omplications associated with natural PCR inhibitors.J. Clin. Lab. Anal . 11 :87-93. (C) 1997 Wiley-Liss, Inc.