PRECISE QUANTITATION OF CHLORAMPHENICOL ACETYL TRANSFERASE REPORTER MESSENGER-RNA 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. coi l 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 compet itive 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 primer s used are both located in the CAT coding sequence at equal distance f rom the EcoRI site. After amplification, the PCR products from the cDN A to be quantified and from the competitor DNA are discriminated by Ec oRI digestion, followed by separation of the resulting fragments by ag arose gel electrophoresis. DNA amplified from the target cDNA is the o nly DNA digested by EcoRI into two fragments of identical size co-migr ating in an agarose gel. After ethidium bromide staining, comparison o f the intensity of fluorescence of the resulting bands in a competitio n series permits us to estimate precisely the amount of CAT cDNA and t herefore of the mRNA to be quantified.