Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion.

TLDR

The study uses DNA from a patient with a small interstitial deletion on the short arm of the X chromosome. The authors describe a method for specifically cloning DNA fragments missing in patients with chromosomal deletions and propose its extension to other loci, such as tumors with small deletions. The method uses phenol‑accelerated competitive DNA reassociation of a 200‑fold excess of patient DNA with Mbo I‑cleaved DNA from a 49, XXXXY cell line, followed by molecular cloning of the reassociated molecules. Analysis of 81 unique cloned segments identified four (5 %) human DNA fragments absent from the patient’s DNA, localized to three regions near band 21 of the X chromosome, serving as potential linkage markers for the boy’s diseases and as starting points for cloning the associated X‑linked loci.

Abstract

A method that allows the specific cloning of DNA fragments absent from patients homozygous or hemizygous for chromosomal deletions is described. The method involves phenol-accelerated competitive DNA reassociation and subsequent molecular cloning of appropriately reassociated molecules. The deletion DNA sample utilized in the competition was isolated from a patient with a minute interstitial deletion in the short arm of the X chromosome. Sheared DNA isolated from a male child, who was diagnosed as having Duchenne muscular dystrophy, chronic granulomatous disease, and retinitis pigmentosa, was combined in a 200-fold excess with Mbo I-cleaved DNA isolated from a 49, XXXXY human lymphoid cell line, and the mixture was subjected to a phenol-enhanced reassociation technique. Analysis of 81 unique segments derived from cloned reassociated DNA molecules has led to the identification of 4 (5%) human DNA fragments that are absent from the male patient's DNA. The 4 clones were localized, on the basis of hybridization with restriction nuclease-digested genomic DNA from a panel of human and human-rodent hybrid cell lines, into three regions surrounding band 21 of the short arm of the normal human X chromosome. These clones are potential linkage markers for the diseases affecting this boy. Each clone, as well as others obtainable by this approach, may also serve as a starting point in the eventual cloning of these three X-linked-disease loci. Extension of this approach to other loci, including human tumors potentially homozygous for small deletions, should also be possible.