Precise quantitation of chloramphenicol acetyl transferase reporter mRNA by competitive polymerase chain reaction.
A strategy, based on competitive polymerase chain reaction (PCR) after reverse transcription, was developed to quantitate mRNA of the E. coli chloramphenicol acetyl transferase (CAT) gene. Precise quantitation of this rare mRNA is achieved by co-amplification of a constant amount of the complementary DNA (cDNA) with successive dilutions of a competitive DNA template of known concentration and with the use of the same primers. The unique EcoRI site located in the CAT coding sequence has been abolished in the competitive DNA. The two oligonucleotide primers used are both located in the CAT coding sequence at equal distance from the EcoRI site. After amplification, the PCR products from the cDNA to be quantified and from the competitor DNA are discriminated by EcoRI digestion, followed by separation of the resulting fragments by agarose gel electrophoresis. DNA amplified from the target cDNA is the only DNA digested by EcoRI into two fragments of identical size co-migrating in an agarose gel. After ethidium bromide staining, comparison of the intensity of fluorescence of the resulting bands in a competition series permits us to estimate precisely the amount of CAT cDNA and therefore of the mRNA to be quantified.
Greco
A
A
CNRS UMR30, Faculté de Médecine, Lyon, France.
Massé
T
T
Madjar
J J
JJ
D001483:Base Sequence; D001667:Binding, Competitive; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000201:enzymology; D004996:Ethidium; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D013194:Staining and Labeling
D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D015246:Deoxyribonuclease EcoRI; D004996:Ethidium