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. M., Slattery, M. , & 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.
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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|Item Type:||Journal Article|
|Keywords:||protein p53, amino acid sequence, article, cancer diagnosis, dna sequence, gene targeting, oligonucleotide probe, oncogene k ras, point mutation, polymerase chain reaction, Base Sequence, Cystic Fibrosis Transmembrane Conductance Regulator, DNA Mutational Analysis, DNA Primers, DNA-Directed DNA Polymerase, Exons, Genes, p53, Genes, ras, Genetic Screening, Humans, Neoplasms, Sensitivity and Specificity, Sequence Deletion, Taq Polymerase|
|Subjects:||Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > BIOCHEMISTRY AND CELL BIOLOGY (060100)|
|Divisions:||Current > Institutes > Institute of Health and Biomedical Innovation|
|Deposited On:||22 Jul 2011 15:41|
|Last Modified:||25 Jul 2011 13:54|
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