Type-specific amplification of viral DNA using touchdown and hot start PCR.

Two types of JC virus (JCV) are found in infected brain and kidney tissues. A highly reliable PCR assay to determine viral type in tissue is presented. This type-specific system is analogous to allele-specific PCR used to detect point mutations in cellular genes. Specific amplification of two fragments, using four pairs of type-specific primers, is based on a single nucleotide difference at the 3'-ends of the primers. A combination of three conditions in the PCR reaction was required for specificity: 'hot start', a ramped ('touchdown') cycle profile, and a slightly lowered molar concentration of the specific primers and dNTPs. Efficient yield of PCR product is not lost under these conditions, and even the least selective mismatches (C:A and T:G) provided specific amplification. Type-specific restriction enzyme sites within the amplified fragments confirm type designation.