Quantitation of targets for PCR by use of limiting dilution.

TLDR

The study presents a general method to quantify the total number of initial DNA targets in a sample using limiting dilution, PCR, and Poisson statistics. The method employs limiting dilution PCR of a leukemic IgH gene, with an optimized all‑or‑none endpoint, an N‑ras internal control to assess amplifiable genomes and DNA degradation, and a two‑stage amplification to overcome competition with normal IgH sequences. In eight leukemic samples, the method detected about two leukemic IgH targets amid 160,000 competing genomes, demonstrating its ability to quantify initial DNA targets, avoid radioactivity, manage excess competitors, and assess DNA degradation.

Abstract

We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.