Quantitative PCR

Quantitative PCR recently has become a powerful tool in clinical investigations. Its main areas of application have been the assessment of residual disease after treatment of leukemia and lymphoma, the detection of viral nucleic acids, and the detection of gene amplification or deletion and of aneuploidy (1).The application of PCR as a quantitative tool requires the solution of the problem of how to reliably determine the initial amount of target template (T ( o )) from the amount of PCR product (T ( n ) that has accumulated after some number (n) of cycles. The relation between (T ( o )) and (T ( n ) in most instances strongly depends on the PCR conditions. This difficult situation is caused by two main properties of PCR: first, the huge over-all amplification factor makes PCR very sensitive to small variations of the experimental conditions; the second problem is caused by the saturation phenomenon, i.e., the gradual decrease of the amplification efficiency that starts in the later stages of PCR, usually following the accumulation of some threshold amount of product.