Purification of silica-free DNA and characterization of its role in coagulation.
Author(s): Medeiros SK, Zafar N, Liaw PC, Kim PY
Publication: J Thromb Haemost, 2019, Vol. 17, Page 1860-1865
PubMed ID: 31309685 PubMed Review Paper? No
Purpose of Paper
This paper compared the procoagulant activity and polyphosphate contamination in DNA extracted from buffy coats using the QIAamp DNA Blood Mini Kit to DNA extracted from whole blood using the PAXgene Blood DNA Kit or the QuickGene DNA Whole Blood Kit and investigated if DNAse I or corn trypsin inhibitor neutralized the procoagulant activity of the DNA.
Conclusion of Paper
DNA extracted using each of the kits was of comparable size and while procoagulant activity was noted in DNA isolated from whole blood using the PAXgene Blood DNA Kit or the QuickGene DNA Whole Blood Kit, it was attenuated through the addition of DNase 1 or corn trypsin inhibitor. In contrast, DNA extracted from buffy coat using the QIAamp DNA Blood Mini Kit had procoagulant activity that was only attenuated through the addition of corn trypsin inhibitor. Polyphosphate P was undetectable in the majority of DNA specimens but was detected in some DNA specimens extracted from buffy coat using the QIAMP DNA Blood Mini Kit and from whole blood using the PAXgene Kit. The authors conclude that DNA isolated using PAXgene or QuickGene, but not QIAamp, is suitable for functional studies.
Studies
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Study Purpose
This study compared the procoagulant activity and polyphosphate contamination in DNA extracted from buffy coats using the QIAamp DNA Blood Mini Kit to DNA extracted from whole blood using the PAXgene Blood DNA Kit or the QuickGene DNA Whole Blood Kit and investigated if DNAse I or corn trypsin inhibitor could neutralize the procoagulant activity of the DNA. Buffy coats were obtained from citrated blood specimens from 3-6 healthy donors by an unspecified method. DNA was isolated from buffy coats using the QIAamp DNA Blood Mini Kit. DNA was extracted from blood collected in PAXgene Blood DNA tubes from 3-6 healthy volunteers using the PAXgene Blood DNA Kit and resuspended in water. DNA was extracted from trisodium citrate blood from 3-6 volunteers using the QuickGene DNA Whole Blood Kit L for DNA isolation from large volume samples by loading each column four times with cell lysate and eluting with water. DNA purity was evaluated using gel electrophoresis and specimens were stored at -80°C. Thrombin-like activity of extracted DNA and calf thymus DNA was evaluated using a chromogenic assay based on the cleavage of S‐2238 substrate. Thrombin generation was evaluated fluorometrically in the presence of differing amounts of PAXgene DNA (0, 5, 10, and 20 µg/mL) using the Technothrombin TGA substrate mixture. Clot formation was monitored turbidometrically in the presence of varying amounts of DNA extracted using each of the kits. Polyphosphate was detected by electrophoresis with DNA extracted using each of the kits. Calf thymus DNA was used as a control in each experiment.
Summary of Findings:
DNA extracted using each of the kits was of comparable size and only calf thymus DNA had thrombin-like activity. The addition of PAXgene-isolated DNA increased the lag time in the thrombin generation assay in a dose-dependent manner that was partially attenuated by the addition of DNAse 1. Thrombin generation was not measurable in DNA extracted from whole blood using the QuickGene DNA Whole Blood Kit due to precipitation of the substrate. Each of the DNA clot times was shortened in the presence of extracted DNA with the largest effect noted for DNA extracted from buffy coats using the QIAamp DNA Blood Mini Kit, followed by DNA extracted from whole blood using the PAXgene Kit. Importantly, the addition of DNAse 1 returned clot times to control levels when DNA was extracted from whole blood using the PAXgene Kit or the QuickGene DNA Whole Blood Kit, but the clot time remained shortened for DNA extracted from buffy coats using QIAamp DNA Blood Mini Kit after the addition of DNAse 1 (P<0.05). The addition of corn trypsin inhibitor attenuated the effects of DNA on clot time, regardless of DNA source/extraction method. Polyphosphate P was undetectable in the majority of DNA specimens but it was detectable in some DNA specimens extracted from buffy coat using the QIAamp DNA Blood Mini Kit and blood using the PAXgene Kit.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Electrophoresis Protein Fluorometry Protein Spectrophotometry Small molecule 1D/2D gels Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp DNA Blood mini kit
PAXgene Blood DNA kit
QuickGene DNA whole blood kit large