PCR Direct from Whole Blood
PCR-based genetic testing is well-established and a fast-growing discipline. Current protocols rely on a DNA extraction step, as wild-type DNA polymerases are completely inhibited by less than 0.2% whole human blood. The mechanism of inhibition is not clearly understood, but proposed to be as a result of the inactivation of the DNA polymerase by multiple inhibitors (e.g. hemoglobin, IgG, lactoferrin, proteases, anticoagulants and salts) and inaccessibility of the template and/or primers in the crude sample.
Using a molecular evolution strategy, Kapa Biosystems has engineered the first DNA polymerase designed specifically for the efficient amplification of DNA fragments directly from whole EDTA blood. The enzyme is available in KAPA Blood PCR Mix A or B, two optimized, ready-to-use 2x formats containing all components required for Whole Blood PCR, except for primers and template. KAPA Blood PCR Kits offer the following benefits for genetic testing and other PCR-based applications that use DNA extracted from blood as template:
- Reduced costs, turnaround times and risk of contamination by eliminating the need for DNA extraction or sample pretreatment.
- Versatility with respect to sample collection. Using KAPA Blood PCR Mixes, DNA may be amplified directly from whole blood collected in EDTA anticoagulant tubes, or on Whatman 903® Specimen Collection (“Guthrie”) Cards, Whatman FTA® Elute Cards, or regular filter paper.
- Versatility with respect to amplicon type. Amplicons up to 3.5 kb with a GC-content <65%, and amplicons <1 kb with a GC content >65% have been amplified successfully using KAPA Blood PCR Kits.
- Compatibility with existing assays and workflows. KAPA Blood PCR Kit may be used in single- or multiplex PCR assays employing standard or fluorescently labelled primers, is suitable for SNP detection using SNaPshot® technology (Applied Biosystems), RFLP analysis and direct sequencing, and paternity testing using the PowerPlex® 16 System (Promega). Reaction products may be analysed by gel electrophoresis, capillary electrophoresis (e.g. using genetic analyzers), dHPLC and on electrophoresis chips.