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.
Ault
G S
GS
Laboratory of Experimental Neuropathology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Ryschkewitsch
C F
CF
Stoner
G L
GL
D001483:Base Sequence; D001921:Brain / Q000382:microbiology; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics; D002141:Encephalitis Virus, California / Q000145:classification* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D004279:DNA, Viral; D004262:DNA Restriction Enzymes