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The effect of storage on (1) amplifiability of nucleic acid (present at low level) and (2) properties of whole blood polymerase chain reaction (PCR) inhibitors (present at high levels) in Guthrie card bloodspots was evaluated. Natural PCR inhibitors (protein, hemoglobin, iron) were selectively eluted from Guthrie cards (1 to 30 mo storage) under nondenaturing conditions and quantitated. The PCR was performed by direct amplification. It was found that PCR inhibitors become increasingly resistant to elution ("fixed") over time. For example, 600 micrograms protein, 1.87 au hemoglobin, and 374 ng iron were solubilized from 1 mo bloodspots. In contrast, only 137 micrograms protein (22 percent), 0.34 au hemoglobin (18 percent), and 147 ng iron (39 percent) were solubilized from 30 mo bloodspots. Fixation does not result from excessive desiccation since bloodspot weight 2.20 mg +/- 0.21 (1 mo) and 1.92 mg +/- 0.31 (30 mo) was not significantly changed (p0.05). The majority of protein was characterized as albumin, and two rbc metal-containing proteins, carbonic anhydrase and hemoglobin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite the presence of "fixed" PCR inhibitors, it was found that bloodspots stored 1 to 30 mo could 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 low yield of deoxyribonucleic acid (DNA) reported for microextraction methods. |
