期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1994
卷号：91
期号：12
页码：5377-5381
DOI：10.1073/pnas.91.12.5377
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The identification of transcripts from large genomic regions cloned in yeast artificial chromosomes (YACs) or cosmids continues to be a critical and often rate-limiting step in positional cloning of human disease genes. We have developed a PCR-based method for rapid and efficient generation of probes from YACs or cosmids that can be used for cDNA library screening. The method, which we call island rescue PCR (IRP), is based upon the observation that the 5' ends of many genes are associated with (G+C)-rich regions called CpG islands. In IRP, the YAC of interest is digested with a restriction enzyme that recognizes sequences of high CpG content, and vectorette linkers are ligated to the cleaved ends. The PCR is used to amplify the region extending from the cleaved restriction enzyme site to the nearest SINE (Alu) repeat. In many cases this product contains sequences from the 5' end of the associated gene. cDNA clones isolated with these products are then verified by mapping them back to the original YAC. The method allows rapid screening of > 500 kb of human genomic insert in one experiment, is tolerant of contaminating yeast sequences, and can also be applied to cosmid pools. In a control experiment, the method was able to identify cDNA clones for the neurofibromatosis type 1 (NF1) gene using a probe generated from a YAC in the region. Application of IRP has yielded nine other genes from YACs isolated from chromosome locations 4p16.3 and 17q21.
