Purification of silica‐free DNA and characterization of its role in coagulation
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BACKGROUND: Although extracellular DNA has been reported to activate coagulation, its direct effects and consequent interpretations have recently been questioned because of silica and polyphosphate (polyP) contaminations when DNA is isolated using common silica-based kits. OBJECTIVES: To identify and characterize alternative methods of isolating DNA that is free of silica with functionally undetectable levels of polyP. METHODS: DNA was isolated from the whole blood or buffy coat using three different DNA isolation kits: (a) the silica-based QIAGEN QIAMP DNA Blood mini kit (silica-DNA), (b) the non-silica-based QIAGEN PAXgene Blood DNA kit (PAX-DNA), and (c) the non-silica-based QuickGene DNA whole blood kit large (DBL-DNA). The procoagulant properties of DNA were assessed by thrombin generation and plasma clotting assays. A polyP detection assay was used to detect polyP contamination. RESULTS AND CONCLUSIONS: Unlike the isolated DNA, commercially available calf thymus DNA contains thrombinlike amidolytic activity. The PAX-DNA and DBL-DNA did not contain silica nor functionally detectable polyP as contaminants. Both PAX- and DBL-DNA were procoagulant in a dose-dependent manner, which is neutralized with deoxyribonuclease I (DNase I). Thus, we recommend the use of PAX-DNA or DBL-DNA for functional studies to investigate the role of extracellular DNA.
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