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 (PCR) is a convenient alternative to the more conventional methods of random primer-labeling and nick translation. PCR requires less template and allows the synthesis of radiolabeled probes from specific sequences contained within cloning vectors and genomic DNA. Under nucleotide imbalance conditions where the concentration of the radiolabeled nucleotide was 0.825 microM and the other dNTPs were each > 25 microM, amplification by Taq DNA polymerase was inhibited. Reducing the concentrations of the unlabeled dNTPs resulted in greater yields of amplification product with maximal yield obtained when the concentration of three unlabeled nucleotides was two- to eightfold higher than that of the limiting labeled nucleotide. When we utilized this amplification method for synthesis of an 800-bp glyceraldehyde-3-phosphate (GAPDH) dehydrogenase probe, 87% of the added [32P]dCTP was incorporated into amplification product. Application of this method for synthesis of high specific activity probes ( > 4 x 10(9) cpm/micrograms) up to 2.6 kb in length is demonstrated and utility of the 800-bp GAPDH probe for hybridization to Northern blots for detection of GAPDH mRNA is presented.
Mertz
L M
LM
Life Technologies, Incorporated, Gaithersburg, Maryland 20884.
Rashtchian
A
A
D001483:Base Sequence; D015152:Blotting, Northern; D015342:DNA Probes; D005784:Gene Amplification; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis
D015342:DNA Probes; D009711:Nucleotides; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases