Prevention of false results from preferential PCR amplification or VNTR alleles

In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alleles is the prevailing method for forensic identification, the fidelity of amplification of heterozygous VNTR alleles with large disparities in length needs to be carefully examined. Reports in the literature and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of genomic DNA, the number of amplification cycles and the length of alleles amplified. Two small (< 1 kb) hypervariable VNTRs (Apo B and HVR-Ig) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the heterozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and therefore, led to the false results; and the allelic fragment of PCR products was preferentially lost after only 2 cycles of overamplification. We also further established the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our routine forensic screening this relationship has been successfully applied to determine the optimal number of amplification cycles and to avoid the allelic drop-out problem and achieve fidelity of PCR-VNTR amplification. It has also been used to investigate forensic casework.