THE EFFECT OF STORAGE ON GUTHRIE CARDS - IMPLICATIONS FOR DEOXYRIBONUCLEIC-ACID AMPLIFICATION

The effect of storage on (1) amplifiability of nucleic acid (present a t low level) and (2) propel-ties of whole blood polymerase chain react ion (PCR) inhibitors (present at high levels) in Guthrie card bloodspo ts was evaluated. Natural PCR inhibitors (protein, hemoglobin, iron) w ere selectively eluted from Guthrie cards (1 to 30 mo storage) under n ondenaturing conditions and quantitated. The PCR was performed by dire ct amplification. It was found that PCR inhibitors become increasingly resistant to elution (''fixed'') over time. For example, 600 mu g pro tein, 1.87 au hemoglobin, and 374 ng iron were solubilized from 1 mo b loodspots. In contrast, only 137 mu g protein (22 percent), 0.34 au he moglobin (18 percent), and 147 ng iron (39 percent) were solubilized f rom 30 mo bloodspots. Fixation does not result from excessive desiccat ion since bloodspot weight 2.20 mg +/- 0.21 (1 mo) and 1.92 mg +/- 0.3 1 (30 mo) was not significantly changed(p > 0.05). The majority of pro tein was characterized as albumin, and two rbe metal-containing protei ns, carbonic anhydrase and hemoglobin by sodium dodecyl sulfate-polyac rylamide gel electrophoresis (SDS-PAGE). Despite the presence of ''fix ed'' PCR inhibitors, it was found that bloodspots stored 1 to 30 mo co uld be amplified for two regions (98 bp and 491 bp amplicons) encoding the Delta F508 cystic fibrosis mutation. It is suggested that nucleic acid also becomes ''fixed'' to the filter paper matrix and accounts, in part, for the ability to amplify Guthrie cards by direct PCR and lo w yield of deoxyribonucleic acid (DNA) reported for microextraction me thods.