Mutation detection in large genes: Use of overlapping PCR fragments for {open_quotes}double{close_quotes}screening of the APC gene and a PCR modification to concatenate multiple exons
- Univ. of Cincinnati College of Medicine, Cincinnati, OH (United States)
- Univ. of Utah, Salt Lake City, UT (United States)
We report developments of earlier work which demonstrated the suitability of PCR-cloning in the 5{prime} terminus of IacZ for the detection of frameshift and nonsense mutations of in APC, the gene which predisposes carriers to adenomatous polyposis coli and, ultimately, colon cancer. A test set of clinically diagnosed APC patients and their unaffected relatives is being used to test a combination of multiplex PCR of several overlapping segments of coding sequence and a lethal negative selection against in-frame, i.e., wild type, clones. This modification will balance the strigency of the negative selection with the low-level readthrough, frameshifting and/or re-initiation of mutant clones, such that high-level {beta}-galactosidase production results in death, while a low level allows discrimination between APC mutant insertions and insert-free vector transformants. The same patient base is also serving as a test set for a PCR-based cloning technique that allows the concatenation of two exons into a single synthetic fragment by skipping over the intervening intron, bypassing the need for single exon analysis or mRNA/cDNA preparation. This technique is based on the design of primers with homology to intron sequences immediately adjacent to the exon sequences of interest, maintaining the reading frame, and adjusting for in-frame termination codons by base-substitutions. The conditions necessary for the concatenation of a large number of exons in a two-step PCR protocol are under investigation. These methods have advantages over other rapid mutation-detection systems by being (1) capable of detecting previously unidentified mutations, (2) non gel-based and (3) non-radioactive.
- OSTI ID:
- 134345
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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