QUANTITATIVE-ANALYSIS OF POLYMERASE CHAIN-REACTION PRODUCTS BY DOT-BLOT

Quantitative analysis of polymerase chain reaction (PCR) products is u sually accomplished by gel electrophoresis and Southern blotting. We h ave developed an alternative technique that allows PCR products to be directly quantitated from unfractionated samples. The PCR was used to amplify genomic (endogenous) DNA sequences (actin) and exogenous DNA ( herpes simplex virus-1 (HSV-1) ribonucleotide reductase) isolated from the trigeminal ganglia of rabbits to demonstrate the dot blot method of PCR product analysis. Two primer pairs (actin and ribonucleotide re ductase) were coamplified, resulting in two different PCR products. Du plicate aliquots of the PCR products were applied to separate nylon me mbranes and hybridized with P-32-labeled oligonucleotide probes. Each radioactive probe was specific for target (HSV-1 DNA) or control (acti n DNA) products. Quantitation using a laser scanning PhosphorImager an d ImageQuant software demonstrated that the dot blot method can be use d to rapidly analyze a large number of PCR samples. (C) 1996 Academic Press, Inc.