Abstract
A major advance in physical mapping of the human genome was the development of yeast artificial chromosome (YAC) vectors (1). This has enabled the cloning of pieces of DNA several hundred kilobases in length (2). The availability of such large cloned genomic DNA fragments means that by ordering a series of overlapping YAC clones, a contiguous stretch of DNA, several megabases in length, can be isolated around a genomic region of interest (e.g., the region of a chromosome linked to a particular disease gene). The successful isolation of terminal sequences of a given YAC can be very useful in assembling an ordered “contig” of YAC clones. Such terminal clones may be used directly as hybridization probes or sequenced and used to generate sequence tagged sites (STSs) to identify overlaps between, and isolate other, members of the contig. Several methods have been used to this end, including PCR with vector-specific primers in combination with primers designed either for repetitive elements, such as Alu sequences (3), or in combination with random nonspecific primers (4). However, these techniques rely on a suitable repetitive element or random primer sequence occurring close enough to the end of the YAC so as to be amplified by PCR. Furthermore, probes isolated in this manner may well contain highly repetitive sequences that, if unsuccessfully blocked, will increase nonspecific signal in any subsequent hybridization procedures (5).
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References
Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806–812.
Cohen, D., Chumakov, I., and Weissenbach, J. (1993) A first-generation physical map of the human genome. Nature 366, 698–701.
Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., Ramirez-Sob, R., Webster, T. D., Ledbetter, D. H., and Caskey, C. T. (1989) Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. USA 86, 6686–6690.
Wesley, C. S., Myers, M. P., and Young, M. W. (1994) Rapid sequential walking from termini of cosmid, P1 and YAC inserts. Nucleic. Acids Res. 22, 538–539.
Cole, C. G., Patel, K., Shipley, J., Sheer, D., Bobrow, M., Bentley, D. R., and Dunham, I. (1992) Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labelled via linear amplification. Genomics 14, 931–938.
Riley, J., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., Anand, R., Smith, J. C., and Markham, A. F. (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 18, 2887–2890.
Valdes, J. M., Tagle, D. A., and Collins, F. S. (1994) Island rescue PCR: A rapid and efficient method for isolating transcribed sequences from yeast artificial chromosomes and cosmids. Proc. Natl. Acad. Sci. USA 91, 5377–5381.
Larsen, F., Gundersen, G., Lopez, R., and Prydz, H. (1992) CpG islands as gene markers in the human genome. Genomics 13, 1095–1107.
Lovett, M., Kere, J., and Hinton, L. (1991) Direct selection: A method for isolation of cDNAs encoded by large genomic regions. Proc. Natl. Acad. Sci. USA 88, 9628–9632.
Buckler, A. J., Chang, D. D., Graw, S. L., Brook, J. D., Haber, D. A., Sharp, P. A., and Housman, D. E. (1991) Exon amplification: A strategy to isolate mammalian genes based on RNA splicing. Proc. Natl. Acad. Sci. USA 88, 4005–4009.
Elvin, P., Slynn, G., Black, D., Graham, A., Butler, R., Riley, J., Anand, R., and Markham, A. F. (1990) Isolation of cDNA clones using yeast artificial chromosome probes. Nucleic Acids Res. 18, 3913–3917.
Roberts, R. G., Coffey, A. J., Bobrow, M., and Bentley, D. R. (1993) Exon structure of the human dystrophin gene. Genomics 16, 536–538.
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© 1996 Humana Press Inc., Totowa, NJ
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McAleer, M.A., Coffey, A., Dunham, I. (1996). DNA Rescue by the Vectorette Method. In: Rapley, R. (eds) PCR Sequencing Protocols. Methods in Molecular Biology™, vol 65. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-344-9:201
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DOI: https://doi.org/10.1385/0-89603-344-9:201
Publisher Name: Springer, Totowa, NJ
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