PCR Bias Toward the Wild-Type k-ras and p53 Sequences: Implications for PCR Detection of Mutations and Cancer Diagnosis
Description
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 on 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 k-ras 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 polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin 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.
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ID Code: | 43505 | ||
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Item Type: | Contribution to Journal (Journal Article) | ||
Refereed: | Yes | ||
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Measurements or Duration: | 8 pages | ||
ISSN: | 0736-6205 | ||
Pure ID: | 34437364 | ||
Divisions: | Past > QUT Faculties & Divisions > Faculty of Health Past > QUT Faculties & Divisions > Faculty of Science and Technology Past > QUT Faculties & Divisions > Science & Engineering Faculty Current > Research Centres > Australian Research Centre for Aerospace Automation |
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Copyright Owner: | Bio-Techniques | ||
Copyright Statement: | This work is covered by copyright. Unless the document is being made available under a Creative Commons Licence, you must assume that re-use is limited to personal use and that permission from the copyright owner must be obtained for all other uses. If the document is available under a Creative Commons License (or other specified license) then refer to the Licence for details of permitted re-use. It is a condition of access that users recognise and abide by the legal requirements associated with these rights. If you believe that this work infringes copyright please provide details by email to qut.copyright@qut.edu.au | ||
Deposited On: | 22 Jul 2011 05:41 | ||
Last Modified: | 03 Mar 2024 17:12 |
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