Methods for identifying genomic equivalent markers and their use in quantitating cells and polynucleotide sequences therein

Abstract

Methods for identifying genetic sequences useful as genomic equivalent markers for organisms are described. The method involves determining the ratio of the absolute number of copies of wild type and mutant amplicons in a number of samples from organisms heterozygous for the mutation. After establishing the number of copies of a particular genetic sequence per genome, the sequence may be used as a measure of the number of genomes per sample, in order to normalize the analysis of another target sequence to abundance per cell. By way of example, the CCR5 gene was shown to be present at 2 copies per genome, and used to measure the number of copies per cell of HIV-1 provirions, human herpesvirus-8, and &agr; deletion circles, a measure of recent thymic emigrants for assessing immune reconstitution. The genomic equivalent marker may be use to identify other genomic equivalent markers based on their copy number in proportion to a previously established marker; by way of example the copy number of the &bgr;-actin gene was found to be 16 copies per genome. The genomic equivalent marker may also be use to determine number of cells in a sample, such as from a tissue sample.