PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis
Barnard, R., Futo, V., Pecheniuk, N., Slattery, M., and Walsh, T. (1998) PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis. BioTechniques, 25 (4). pp. 684-691.
PDF (Published Version)
- Published Version
Restricted to Repository staff only
PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work factor IX suggests that this assumption is invalid for one case of near-sequence identity To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify, wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For kras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V cystic fibrosis trans membrane conductance regulator and pro thrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.
|Item Type:||Article (Refereed Research - C1)|
|Keywords:||polymerase chain-reaction; colorectal tumors; amplification|
|Date Deposited:||02 Sep 2010 05:50|
|FoR Codes:||11 MEDICAL AND HEALTH SCIENCES > 1112 Oncology and Carcinogenesis > 111202 Cancer Diagnosis @ 80%
11 MEDICAL AND HEALTH SCIENCES > 1112 Oncology and Carcinogenesis > 111207 Molecular Targets @ 15%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060499 Genetics not elsewhere classified @ 5%
|SEO Codes:||97 EXPANDING KNOWLEDGE > 970111 Expanding Knowledge in the Medical and Health Sciences @ 60%
97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 10%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920102 Cancer and Related Disorders @ 30%