Rapid isolation of cDNA by hybridization (geneybacterial artificial chromosomeyexon trappingysequencing)

The isolation of genes from a given genomic region can be a rate-limiting step in the discovery of disease genes. We describe an approach to the isolation of cDNAs that have sequences in common with large genomic clones such as bacterial artificial chromosomes. We applied this method to loci both amplified and deleted in cancer, illustrating its usage in the identification of both oncogenes and tumor suppressor genes, respectively. The method, called rapid isolation of cDNAs by hybridization (RICH), depends on solution hybridization, enzymatic modification, and amplificationyselection of sequences present in both cDNA populations and the genomic clones. The method should facilitate the development of transcription maps for large genomic clones, possibly even yeast artificial chromosomes. Powerful methods have facilitated the localization of disease genes to regions of the genome. Typically, candidate regions are contained on large yeast or bacterial cloning vectors, and these vectors must be searched assiduously by various means for candidate genes. This step has often proved to be an obstacle in gene discovery. The problem has been attacked in roughly three ways: by hybridization (1–3), sequence analysis (4, 5), and exon trapping methods (6–8). Each method has its own particular advantages, but no current method is without serious problems. We present herein a method we call rapid isolation of cDNAs by hybridization (RICH) based on the identification of sequences in common between a cDNA library and a large clone of genomic DNA. The method selects and amplifies those restriction endonuclease fragments of cDNAs that hybridize precisely at one end to the end of a similarly cleaved genomic DNA fragment. Before hybridization, the cDNA and genomic fragments are modified with different adaptors. Those cDNAs that form hybrids with genomic DNA at at least one end are ligated to a ‘‘selection adaptor’’ that is complementary to the genomic adaptor and contains an additional sequence complementary to an RNA polymerase site. Such cDNAs can be selectively amplified by successive treatments with RNA and DNA polymerases. We illustrate the basic method with two series of experiments: (i) a search for transcripts from the c-MYC locus in cDNAs from a breast cancer cell line and (ii) a search for transcripts from the PTEN tumor suppressor locus in cDNAs from normal breast tissue. Although the method is complex, in that many different enzymes (restriction endonucleases, various DNA ligases, RNA polymerase, various DNA endoand exonucleases, reverse transcriptase, and various DNA polymerases) are used, they are all robust enzymes that are readily available. Only 3 days are required to yield candidates for further study. MATERIALS AND METHODS Materials. The oligonucleotides which were synthesized for this research are listed in Table 1 and obtained from Biosynthesis (Lewisville, TX). P1 clone 8001 (Genome Systems, St. Louis) is an 80-kb genomic clone that contains the exons 1–3 of the c-MYC gene. A bacterial artificial chromosome (BAC) clone, 60C5, containing exons 4–9 of PTEN (containing nucleotides 1,244–2,246 of the published cDNA, GenBank accession no. U92436) was obtained from Genome Systems. The plasmid pUC18, digested with BamHI and treated with bacterial alkaline phosphatase was supplied by Amersham. pCR-Script SK(1) and Epicurian Coli XL2-Blue cells were obtained from Stratagene. SKBr3 is a breast cancer cell line from which the poly(A)1 RNA was extracted with the FastTrack kit (Invitrogen). cDNA was synthesized from the poly(A)1 RNA of SKBr3 and commercially available poly(A)1 RNA of mammary gland tissue (CLONTECH) by using the Copy kit (Invitrogen). The Megascript kit was from Ambion (Austin, TX). The enzymes and their specific buffers used in RICH were obtained from the following suppliers: Sau3AI and T4 DNA ligase from New England Biolabs; Stoffel fragments, AmpliTaq, and AmpliTaq Gold from Perkin–Elmer; Ampligase from Epicentre Technologies (Madison, WI); Pfu DNA Polymerase and RNase-free Dnase from Stratagene; and l exonuclease from Amersham. Glycogen was obtained from Boeringer Mannheim. GeneQuant G-50, S-300HR, and S-400HR columns and Sephaglas BandPrep kit were obtained from Amersham. RNase-free water was supplied by Ambion. Maxi prep kit was supplied by Qiagen (Hilden, Germany). Phenol was prepared as described elsewhere (9). RICH Standard Protocol. To facilitate description of the RICH protocol, we have broken up the procedure into discrete units, labeled those units with an alphabetic letter, and assigned that letter both to the procedure and its final product (Fig. 1). A. Preparation of genomic DNA. For the preparation of genomic DNA, 1 mg of BAC DNA is digested with 20 units of Sau3AI. The digests are purified by phenolychloroform extraction and ethanol precipitation. The whole digests are mixed with 100 pmol of pR-24 and 100 pmol of R-12 oligonucleotides (Table 1) in 20 ml of 13 T4 DNA ligase buffer. The double-strand cDNA and the oligonucleotides are heated at 65°C for 5 min, annealed by cooling down the mixture to 4°C gradually, and then ligated by overnight incubation with 2,000 units of T4 DNA ligase at 11°C. The excess oligonucleotides are removed from the ligated fragments with S-400HR columns. B. Preparation of selection adaptor. The selection adaptor is synthesized, phosphorylated at its 59end, and purified by PAGE. The working concentration of the adaptor is 50 nM. The 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. © 1998 by The National Academy of Sciences 0027-8424y98y953764-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: RICH, rapid isolation of cDNA by hybridization; BAC, bacterial artificial chromosome; RIGHT, rapid isolation of genes by hybridization to transcripts. *To whom reprint requests should be addressed. e-mail: wigler@ cshl.org.