Efficient Production of Internal Standard DNA for Quantitative PCR Using an Automated Sequencer

摘要

Several quantitative or semi-quantitative polymerase chain reaction (PCR) methods have been described recently. Usually, in these techniques, the quantification involves the coamplification of an internal control together with the sequence of interest(1,3,11). More recently two groups reported independently a fluorescence-based quantitative PCR method where the internal standard and the target DNAs were separated and detected by means of an automated sequencer (6-8). One major advantage of this method is that the standard and the target DNA could differ by a few base pairs. Therefore, standard molecules that were quasiidentical to the target DNA could be designed, and both species could be co-amplified with the same efficiencies. Quantification could be performed under those conditions with very good accuracy (6). However, the construction of such internal standards, which involved several steps of cloning or PCR (7,8), was rather time-consuming, laborious and had to be done for every single geneto be assayed.