Elimination of non-viable 6-thioguanine-sensitive T cells from viable T cells prior to PCR analysis

The study of T cell clones at the genomic level is expanding our understand ing of their role in diseases such as rheumatoid arthritis (RA) and multipl e sclerosis (MS). We have been carrying out genotypic analysis by PCR of hy poxanthine phosphoribosyltransferase (hprt) mutations in these cells. Mutan t T cells in the population can be cloned on the basis of their resistance to the cytotoxic drug, 6-thioguanine-(6-TG). A difficulty is that the major ity of primary human T cells are capable of only limited growth ex vivo, ev en in the presence of 'feeder' cells. PCR analysis of DNA from such clones is made difficult by the limited number of viable mutant (drug-resistant) T cells and the large number of dead (drug-sensitive) mononuclear cells and feeder cells. DNA from the 'dead' cells remains sufficiently intact for man y weeks in culture and can represent a significant source of background in PCR analysis. Here we describe a method employing hypotonic shock and micro coccal nuclease that reliably eliminates non-viable 6-TG-sensitive cells, a llowing the study of the hprt gene in < 200 T cells by PCR. (C) 1999 Elsevi er Science B.V. All rights reserved.