title: Quantitation of targets for PCR by use of limiting dilution.
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.
authors.affiliation: Department of Hematology, Flinders Medical Center, Bedford Park, South Australia.
authors.forename: P J
authors.initials: PJ
authors.lastname: Sykes
authors.forename: S H
authors.initials: SH
authors.lastname: Neoh
authors.forename: M J
authors.initials: MJ
authors.lastname: Brisco
authors.forename: E
authors.initials: E
authors.lastname: Hughes
authors.forename: J
authors.initials: J
authors.lastname: Condon
authors.forename: A A
authors.initials: AA
authors.lastname: Morley
mesh_terms: D001483:Base Sequence; D004273:DNA, Neoplasm / Q000032:analysis*; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D011905:Genes, ras; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D013698:Templates, Genetic
chemical_list: D004273:DNA, Neoplasm; D007143:Immunoglobulin Heavy Chains