The generation of radiolabeled DNA and RNA probes with polymerase chain reaction.
By including a radioactive triphosphate during polymerase chain reaction (PCR), probes of very high specific activity can be generated. The advantages of PCR labeling include (1) uniform labeling with a specific activity of 5 X 10(9) cpm/micrograms or higher (sensitivity of detection: 0.028 pg of target DNA per 24 h); (2) ease of regulation of both the specific activity and the amount of labeled probe produced; (3) efficient labeling of fragments less than 500 bp; (4) efficient incorporation over a wide range of input DNA template; (5) labeling with subnanogram amounts of input DNA; and (6) direct labeling of genomic DNA. The minimal amount of input DNA allows a virtually unlimited number of PCR labeling reactions to be performed on DNA generated by one amplification under the previously described nonlabeling conditions. This obviates the need for CsCl gradients or other large scale methods of DNA preparation. The above advantages except for the very high specific activity can also be achieved by transcript labeling after an amplification where one or both of PCR primers contain a phage promoter sequence.
Schowalter
D B
DB
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Sommer
S S
SS
D015139:Blotting, Southern; D015342:DNA Probes / Q000138:chemical synthesis*; D004259:DNA-Directed DNA Polymerase; D010761:Phosphorus Radioisotopes; D015347:RNA Probes / Q000138:chemical synthesis*; D019914:Taq Polymerase; D014158:Transcription, Genetic
D015342:DNA Probes; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase