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Long-distance PCR of VNTR at the D17S74 (CMM86) locus.

We have successfully amplified D17S74 (CMM86) alleles by a long-distance polymerase chain reaction (PCR) using TaKaRa Ex Taq (a Taq DNA polymerase with a 3'-exonuclease activity) and Perfect Match Polymerase Enhancer (a special polymerase enhancer). We adopted a hot-start technique with TaqStart antibody. Because of the high guanine content (60%) in D17S74 alleles, removal of K+ from the buffers was quite effective. The use of K(+)-free buffers reduces premature chain termination in G-rich regions, thereby facilitating amplification of targets containing such sequences. The 17 alleles amplified from DNA samples of 72 unrelated Japanese subjects ranged from 1.05 to 3.5 kb, with a heterozygosity of 92%. PCR amplification of D17S74 alleles makes their detection simpler than by conventional Southern blotting, and increases the practical utility of the locus.