AN EVALUATION OF COMPETITOR TYPE AND SIZE FOR USE IN THE DETERMINATION OF MESSENGER-RNA BY COMPETITIVE PCR

The technique of competitive PCR for measuring mRNA is used widely. Se veral variations of the method have been reported. We have evaluated s ome of the commonly used competitor types as part of our study into ex pression of the androgen receptor (Ah). These included mutant, intron, deletion construct, and nonhomologous competitors, which were assesse d with an emphasis on their ability to amplify the target with the sam e efficiency, as well as their capacity to form heteroduplexes with it . The effect of competitor size on amplification efficiency was also i nvestigated. We found that the use of a common primer set did not guar antee equal amplification efficiencies among DNAs sharing the same pri mer sequences. For the competitors evaluated In this study, sequence l ength was the major determinant of amplification efficiency. The longe st competitors were amplified with the least efficiency. Differences i n amplification efficiencies were corrected for by standardizing the c ompetitor against the target. Constructing competitors of different si zes to the target may not eliminate heteroduplex formation when they s hare common sequence with the target as with the intron and deletion t ype competitors. Such heteroduplexes may interfere with the analysis i f they cannot be resolved from both the target and competitor. Use of a mutant competitor constructed by the conversion of one enzyme restri ction site to another produced determinations that were Independent of both heteroduplex formation and cycle number. A method is described f or generating a mutant competitor with a single PCR. The results of th is study show that when competitive PCR Is being used to determine abs olute levels of mRNA, the competitor constructed should be assessed ca refully for its ability to amplify with the same efficiency as the tar get and for its capacity to form heteroduplexes with it.