PCR on Cell Lysates Obtained from Whole Blood Circumvents DNA Isolation

Current methods for PCR use purified genomic DNA, isolated mostly from leukocytes. To avoid time-consuming, and therefore expensive, procedures to purify genomic DNA, methods have been developed that use whole blood or buffy coats for PCR (1)(2)(3)(4)(5). However, extensive washing procedures become necessary to remove hemoglobin and anticoagulants, which inhibit PCR (3)(6)(7). In addition, heat-cool cycles have to be included to liberate DNA from the nucleus (1)(5). Furthermore, PCR protocols are optimal for genomic DNA, but the salt composition of the PCR mixture needs to be readjusted if PCR is performed with whole blood or buffy coats (5)(8). Finally, digestion of PCR products with restriction enzymes may be severely inhibited by anticoagulants (9)(10). We developed a simplified method to overcome these drawbacks. Briefly, red blood cells are first lysed in a large volume, which dilutes the hemoglobin and anticoagulants to an extent that PCR and post-PCR treatments are not inhibited. The white blood cells are then resuspended in a Tris-EDTA (0.67 mmol/L Tris-HCl, 0.07 mmol/L EDTA, pH 7.5) buffer commonly used to store genomic DNA; this eliminates readjustment of the salt composition of the PCR mixture. Finally, because one freeze-thaw cycle disrupts white blood cells into cellular fragments more efficiently than several heat-cool cycles, storage at −20 °C of white blood cells resuspended in Tris-EDTA should yield, after thawing, a cell lysate suitable for PCR. This assumption was tested using cell lysates for apolipoprotein E ( APOE ) genotyping in a PCR protocol optimized for genomic DNA. Anonymized cell lysates were prepared from whole-blood samples collected in Vacutainer Tubes (Becton Dickinson) by venipuncture. One set of samples was collected from 24 consecutive different patients referred for routine clinical chemistry laboratory investigation; these samples …