PREVENTION OF FALSE RESULTS FROM PREFERENTIAL PCR AMPLIFICATION OF VNTR ALLELES

In forensic DNA typing, evidential samples generally involve limited a mounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alle les is the pre-prevailing method for forensic identification, the fide lity of amplification of heterozygous VNTR alleles with large disparit ies in length needs to be carefully examined. Reports in the literatur e and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of g enomic DNA, the number of amplification cycles and the length of allel es amplified. Two small (< 1 kb) hypervariable VNTRs (Ape B and HVR-Ig ) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the het erozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and, therefore, led to false re sults; and the larger allelic fragment of PCR products was preferentia lly lost after only 2 cycles of overamplification. We also further est ablished the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our r outine forensic screening, this relationship has been successfully app lied to determine the optimal number of amplification cycles and so av oid the allelic drop-out problem and achieve fidelity of PCR-VNTR ampl ification. It has also been used to investigate forensic casework.