cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.

Abstract

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. 9623The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

“…Identification of genes in genomic loci associated with human diseases has been greatly facilitated by the development of techniques such as "exon trapping" (1) and cDNA selection (2,3). Direct sequencing of disease loci has also been shown to be one of the most effective methods of gene detection, but it requires significant sequencing capacity (4)(5)(6).…”

Oat–maize chromosome addition lines: A new system for mapping the maize genome

“…The discovery of maize-chromosome retention in oat ''haploids'' after oat ϫ maize crosses and the recovery of stable maize chromosome-addition oat lines (8,9) should allow the development of a system of chromosome analysis similar to that available in mammalian hybrid-cell systems (10)(11)(12). Such a system may be used for gene assignment, isolation of chromosome-specific probes (13), flow sorting (14) and microdissection of chromosomes (15), development of chromosomespecific ''paints'' of fluorochrome-labeled DNA fragments (16)(17)(18), physical mapping, and selective isolation and mapping of cDNAs of a particular chromosome (19,20).…”

Specific isolation of 3′-terminal exons of human genes by exon trapping

“…In theory, trapping of 3'-terminal exons has a number of advantages compared to trapping of internal exons. First, 3'-terminal exons are better defined by the presence of both splicing and polyadenylation signals (14). One of the problems with trapping of internal exons is the activation of cryptic SA We have previously described an exon trapping system, using vector pETV-SD2, to trap internal exons (16 Figure IA.…”

“…Direct cDNA selection (1)(2)(3)(4) and exon trapping (5,6) have proven extremely successful in the cloning of various disease genes, amongst which are the genes responsible for Huntington's disease (7), neurofibromatosis type II (8), X-linked glycerol kinase deficiency (9) and Wilson's disease (10).…”