Isolation of Expressed Sequences from the Chromosome 17q21 BRCA1 Region by Magnetic Bead Capture
Magnetic bead capture of cDNAs utilizes biotin-streptavidin magnetic bead technology to isolate expressed sequences from large genomic regions, resulting in several thousand-fold enrichment of selected cDNAs. The technique allows parallel analysis of several genomic segments of varying complexity. Expressed sequences from a variety of tissue sources can also be identified simultaneously. To evaluate this approach, we have applied it to pools of cosmid clones from the interval on chromosome 17q21 which contains the familial early onset breast cancer gene (BRCA1). We describe the characterization of 9 potentially unique cDNAs which were isolated from one pool of 7 minimally overlapping cosmids representing a subset of the BRCA1 candidate region. Overall, the method is shown to detect a large fraction of coding sequences in cosmid clones. Advantages and limitations of the approach are discussed.
KeywordsFormaldehyde Agarose Immobilization Boiling Nylon
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- 4.D.B. Krizman and S.M. Berget, 3’-Terminal exon trapping: Identification of genes from vertebrate DNA, Focus 15:106.Google Scholar
- 11.D.A. Tagle, M. Swaroop, L. Elmer, J. Valdes, K. Blanchard-McQuate, M. Allard, G. Bates, S. Baxendale, R. Snell, M. MacDonald, J. Gusella, H. Lehrach, and F.S. Collins, Magnetic bead capture of cDNAs: A strategy for isolating expressed sequences encoded within large genomic segments, in “Magnetic Separation in Molecular and Cellular Biology”, M. Uhlen, O. Olsvik, and J. Elingboe, ed., Eaton Publishing Co., In Press.Google Scholar
- 12.T. Maniatis, E.F. Fritsch, and J. Sambrook, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1982).Google Scholar
- 17.J. Simard, J. Feunteun, G. Lenoir, P. Tonin, T. Normand, V. Luu The, A. Vivier, D. Lasko, K. Morgan, G.A. Rouleau, H. Lynch, F. Labrie, and S.A. Narod, Genetic mapping of the breast-ovarian cancer syndrome to a small interval on chromosome 17q12–21: exclusion of candidate genes EDH17B2 and RARA, Hum. Mol. Genet. 2:1193 (1993).PubMedCrossRefGoogle Scholar