Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. Immobilization of Genomic Cosmid or YAC DNA on Nylon Discs. Cosmid DNA (0.2 ng) or YAC DNA (1-2 ng) was digested with EcoPJ, heat-denatured at 950C for 3 min along with 50 ng ofan Hae III digest ofbacteriophage 4X174 DNA, as carrier DNA, and loaded in a 0.5-,ul volume onto 6.25-mm2 nylon discs (Hybond, Amersham), in the presence of 1Ox SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0). The DNA on discs was denatured in 0.5 M NaOH containing 1.5 M NaCl for 5 min. The discs were neutralized with 0.5 M Tris Cl (pH 7.2) containing 1.5 M NaCl for 3 min, either exposed to UV irradiation in a Stratalinker (Stratagene) for 1 min (autolink mode) or baked at 80'C in vacuum for 2 hr, and stored dry at 40C until used.
Quenchers of Nonspecific Hybridization to Nylon Discs. (i)Human chromosome 15 library. DNA from a human chromosome 15 genomic library (15 NSO3, ATCC 57740) was digested with EcoRI and 0.8-to 6-kb (size range) DNA was cloned into the pTZ18 vector (Pharmacia) at the EcoRI site. A total of 3 x 105 recombinants was generated after transformation and ampicillin selection.(ii) Genomic repetitive sequence library (XRLI). XRLI was prepared from the human X chromosome library (LAOXNLO1, ATTC 57750) by digestion of various DNA samples with several single restriction enzymes, pooling, cloning 0.3-to 2-kb (size fraction) DNA in Charon BS vector (22), and probing the resulting library with 32P-labeled total genomic DNA (23). Five hundred positive plaques were processed for isolation of phage DNA and subsequent plasmid preparation (22).(iii) rRNA-specific clones. Plasmid clones containing human rRNA gene EcoRI fragments of 7.3 kb and 5.8 kb cloned into pBR322 at the EcoRI site were kindly provided by D. Ward (Yale University) (24).(iv) Poly(dI)-poly(dC). Material of 724 base pairs (average length; Pharmacia) was used without any further treatment.(v) Yeast DNA. Yeast DNA from strain AB1380, which is the parent host of YAC B30H3, was prepared (20) and sonicated to an average size of 0.7-1 kb. Plasmid DNA from the sources i-iii was sonicated, digested with EcoRI, and treated with 1 unit of mung bean nuclease per pug of DNA at Abbreviations: MHC, major histocompatibility complex; PFGE, pulsed-field gel electrophoresis; YAC, yeast artificial chromosome.TTo whom reprint requests should be addressed.
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Molecular analysis of the human major histocompatibility complex region has identified three largely intact genes (HLA E, HLA 5.4 and HLA 6.0) for nonclassical class I transplantation antigens (1). Although gene transfer results suggest that the relatively non-polymorphic gene HLA 6.0 can potentially encode a class I polypeptide (2), expression of the mRNA from the chromosomal gene is not reported. We isolated the cDNA clone ASR53 for HLA 6.0 (HLA G) from a human fetal eye (11 week) cDNA library. The nucleotide sequence is idential to that
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