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J Virol Methods. 1994 Feb;46(2):145-56.

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

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Laboratory of Experimental Neuropathology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.


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.

[Indexed for MEDLINE]

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