Isolation of DNA clones revealing restriction fragment length polymorphisms in the human genome

Abstract

SummaryA recombinant human DNA library was constructed using pUC 18 as the cloning vector. Plasmid DNA isolated from a small scale culture was used as the hybridization probe; many recombinant clones could be easily tested for their ability to detect restriction fragment length polymorphisms (RFLPs). Forty-five arbitrary single copy DNA fragments were isolated from this library and five clones revealed RFLPs. Probe OS-5 had three alleles and probe OS-7, which detected insertion/deletion polymorphisms, had several alleles.Apart from these clones, two polymorphic DNA fragments were isolated from the pBR322 plasmid library and another two from the Charon 4A phage library. Although only four restriction enzymes were employed to detect polymorphisms, the efficiency of detecting polymorphisms was reasonable.These nine clones will serve as useful markers for linkage studies.

Preclinical detection in Japanese families with myotonic dystrophy using polymorphic DNA markers

“…All patients defined as DM satisfied the criteria for DM proposed by the working group on the molecular defect in myotonic dystrophy (Griggs et al, 1989). DNA was extracted from peripheral white blood cells by using the method of Nishisho et al (1986) and digested with restriction enzyme according to conditions of the suppliers (Takara, Kyoto). Samples were run on 0.7~ agarose gels and blotted on nylon-membrane filter (Hybond-N, Amersham).…”

The zygosity determination of Japanese twins using a minisatellite core probe

“…Heparinized peripheral or umbilical cord blood samples (usnally 3-20 ml) were obtained from eight unrelated Japanese, a Japanese family of three generations, five pairs of twins and a set of triplets. D N A was extracted from white blood cells as described elsewhere (Nishisho et al, 1986). in one individual (Fig.…”

A family case with autosomal-dominantly inherited pituitary dwarfism.

“…Hybridization was carried out at 65°C for 15-20 hr in 1 X Denhardt's solution, 1 M NaCI 50 mM Tris-HC1 pH 7.4, 10 mM EDTA, 0.1% SDS and 0.1 mg/ml denatured sonicated salmone sperm DNA. Filters were washed with 15 mM NaCI/1.5 M sodium citrate/0.1% SDS at 65°C for 90 min with three changes of buffer (Southern 1975;Martial et al 1979;Nishisho et al 1986). Empty sella was found in Case Cases 2 and 3.…”