Amplification of DNA from whole blood.

A method is described for the amplification by PCR of human chromosomal DNA sequences from whole blood samples. Various amounts of blood samples, with either EDTA, citrate, or heparin used as the anticoagulant, have been used to determine optimal PCR conditions for each type of sample. Up to 80% (vol/vol) of whole blood sample is tolerated in PCR with Tac I polymerase. Amplification from whole blood requires the optimization of salt (K § and Mg §247 according to sample volume and type of anticoagulant used. Pretreatment of fresh blood samples to lyse the leukocytes is required for EDTA-treated blood samples and is beneflcient in PCR with heparinand citrate-treated blood samples to obtain maximal amplicon amounts. A satisfactory method of pretreating samples is freeze/thawing. In addition, EDTAtreated blood samples require a heat treatment before PCR for maximal amplicon synthesis. It appears that purification of the DNA is not necessary for any of the whole blood samples analyzed by PCR. Results of amplification reactions from unpurifled hepatitis B virus (HBV) sequences of whole sera samples are shown also. W h o l e blood, plasma, and sera represent, by far, the most commonly used sample types in the diagnostic field. Because PCR inhibitors in blood samples ~ have been described, generally, it is accepted that a careful purification of nucleic acids is required from such samples before PCR analysis can be performed. Classical methods for nucleic acid purification from clinical samples involve reagents and steps like organic solvents, denaturing agents, detergents, centrifugation, and precipitation, ~2'3~ many of which are not compatible with heat-stable DNA polymerases nor desired in future applications with automated PCR systems. Thus, a simple sample preparation method allowing DNA analysis by PCR from whole blood would be valuable. Different groups have succeeded in using small amounts of unpurified blood samples as substrate for PCR. Amplification of DNA from 1 I~1 to 2 i~1 of unpurif ied blood in 100-txl reactions was obtained after a cyclic thermal pretreatment of the samples at 95~ and 55~ ~4~ while it was observed that Tth but not Taq polymerase will amplify DNA from small samples of unpurif ied and untreated blood. ~s~ The work presented here describes conditions under which chromosomal sequences can be amplified from unpurifled blood using EDTA, heparin, or citrate as anticoagulants. Sample volumes constituted 50% or more of the PCR assay.