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The effect of storage on Guthrie cards: implications for deoxyribonucleic acid amplification
G S Makowski1, E L Davis, S M Hopfer
1Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
Insights
Guthrie card bloodspots stored up to 30 months retain amplifiable nucleic acid (DNA) despite increasing "fixed" polymerase chain reaction (PCR) inhibitors, enabling direct PCR amplification.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Guthrie cards are crucial for newborn screening, storing bloodspots for extended periods.
- Storage duration impacts nucleic acid amplifiability and the effectiveness of polymerase chain reaction (PCR) inhibitors.
- Understanding these effects is vital for accurate genetic analysis from stored samples.
Purpose of the Study:
- To evaluate the impact of storage time (1-30 months) on Guthrie card bloodspots.
- To assess changes in nucleic acid amplifiability and PCR inhibitor properties.
- To investigate the
- fixation
- phenomenon of inhibitors and nucleic acids.
Main Methods:
- Selective elution of natural PCR inhibitors (protein, hemoglobin, iron) from Guthrie cards under nondenaturing conditions.
- Quantitation of eluted inhibitors and assessment of their resistance to elution over time.
- Direct amplification PCR to test amplifiability of two gene regions (98 bp and 491 bp) associated with cystic fibrosis.
Main Results:
- PCR inhibitors became increasingly resistant to elution ("fixed") with prolonged storage.
- Significant reduction in solubilized protein, hemoglobin, and iron from older bloodspots (30 months vs. 1 month).
- Despite "fixed" inhibitors, bloodspots stored for 1-30 months remained amplifiable for target gene regions using direct PCR.
Conclusions:
- Nucleic acid (DNA) likely also becomes "fixed" to the Guthrie card matrix over time.
- "Fixed" nucleic acid may contribute to successful direct PCR amplification and explain low DNA yields in microextraction methods.
- Guthrie cards stored for up to 30 months are suitable for direct PCR amplification of specific genetic markers.
Abstract:
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 (p > 0.05). 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.