NUCLEOTIDE IMBALANCE AND POLYMERASE CHAIN-REACTION - EFFECTS ON DNA AMPLIFICATION AND SYNTHESIS OF HIGH SPECIFIC ACTIVITY RADIOLABELED DNA PROBES

Synthesis of radiolabeled DNA probes via polymerase chain reaction (PC R) is a convenient alternative to the more conventional methods of ran dom primer-labeling and nick translation. PCR requires less template a nd allows the synthesis of radiolabeled probes from specific sequences contained within cloning vectors and genomic DNA. Under nucleotide im balance conditions where the concentration of the radiolabeled nucleot ide was 0.825 mu M and the other dNTPs were each >25 mu M, amplificati on by Tag DNA polymerase was inhibited. Reducing the concentrations of the unlabeled dNTPs resulted in greater yields of amplification produ ct with maximal yield obtained when the concentration of three unlabel ed nucleotides was two- to eightfold higher than that of the limiting labeled nucleotide. When we utilized this amplification method for syn thesis of an 800-bp glyceraldehyde-3-phosphate (GAPDH) dehydrogenase p robe, 87% of the added [P-32]dCTP was incorporated into amplification product. Application of this method for synthesis of high specific act ivity probes (>4 X 10(9) cpm/mu g) up to 2.6 kb in length is demonstra ted and utility of the 800-bp GAPDH probe for hybridization to Norther n blots for detection of GAPDH mRNA is presented. (C) 1994 Academic Pr ess, Inc.