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Isolation of expressed sequences by exon trapping and cdna selection using p1 phage clones from chromosomal region 13q12-q13


To identify and isolate the putative BRCA 2 gene P1/P1 derived artificial chromosome (PAC) clones from human chromosome 13ql2-ql3 will be searched for transcribed sequences. By P1/PAC clones large DNA fragments of about 100 kb can be cloned in E. coli where pure DNA can be prepared easily, making them more suitable for exon trapping and cDNA selection than YAC inserts, which often can only poorly separated from yeast host chromosomes. An improved exon trapping protocol suited to analyze genomic fragments from 30 kb-3 Mb will be applied to DNA from single or pooled P1/PAC clones. Isolated exons will be sequenced and used as expressed sequence tags (ESTs) to screen cDNA libraries. P1/PAC DNA will also be used for cDNA selection using pooled cDNA libraries tagged with different linkers for hybridization. Use of exon trapping products for cDNA selection could lead to the identification of low abundant transcripts.


University of Cambridge
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CB2 1QP Cambridge
United Kingdom

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