Isolation of unique sequence human X chromosomal deoxyribonucleic acid

Abstract

Labeled unique sequence human X chromosomal DNA was isolated by hybridization of labeled human DNA with DNA from a human-mouse hybrid cell line, A9/HRBC2-A, which contains a single human chromosome, the X. Homopolymer tails of poly(dA) were added to nick-translated unique sequence human DNA to permit separation of the labeled DNA from unlabeled driver DNA by binding to oligo(dT)-cellulose. Human DNA sequences homologous with mouse DNA were removed from this labeled poly(dA)-tailed human probe by hybridization with excess unlabeled mouse DNA. The labeled human DNA which did not hybridize with mouse DNA was then hybridized with excess unlabeled A9/HRBC2-A DNA, with which only X chromosomal human DNA will hybridize. After hybridization, the labeled human X chromosomal DNA was separated from the unlabeled A9/HRBC2-A DNA by binding to oligo(dT)-cellulose. The purity of the final X chromosomal DNA preparation was > 90%, and the hybridization with mouse DNA was < 2%. When carried out under standard conditions for DNA reassociation, this procedure is complicated by the formation of hybrids between the poly(dA) tails of the probe and T-rich sequences in mouse and human DNA. These imperfectly paired hybrids are less stable than those of unique sequence DNA and can be eliminated by carrying out the hydroxylapatite chromatography at an elevated temperature of 71.degree. C.