Low stringency-PCR (LS-PCR) allows entirely internally standardized DNA quantitation

The quantity of DNA in a sample can be estimated using the polymerase chain reaction (PCR) by comparing the amounts of products that resultfrom the co-amplification of a target sequence and an added internal standard of known concentration that is amplified by the same primers (1-5). In several important applications of competitive PCR, such as the detection of gene amplification in tumors and the quantification of some viral infections, it is the ratio of the number of copies of the target gene to the number of diploid genomes present that is in essence being sought. In achieving this value by competitive PCR there are several points at which error can occur including inter-tube variations in the amount of added standard and exact quantification of the amount of DNA in the sample and variable degrees of sample degradation. We here show that these variables, as well as the need for construction of an internal standard, can be eliminated by using Low Stringency-PCR (LS-PCR) for DNA quantification. In LS-PCR, a specific primer pair is employed under low stringency conditions so that in addition to the specific product a series of other DNA fragments are amplified that result from the low stringency interaction of the primers with other regions of the DNA in the reaction tube (6-8). In the case of the application of LS-PCR to the diagnosis of infection by microorganisms, the low stringency products (LSPs) are invariably amplified from the host DNA due to the relative molecular masses of the genomes of the two organisms. Inspection of our previous data shows that there is competition between the specific product and the LSPs although, in the latter case, the actual sequences being amplified are entirely unrelated to the specific product and indeed may have the same primer at both extremities. We have now found that this competition allows quantification of the specific product in relation to the amount of host DNA present without the need for constructing an internal standard or exact quantitation of the overall amount of DNA in the sample.