PCR Bias Toward the Wild-Type k- ras and p53 Sequences: Implications for PCR Detection of Mutations and Cancer Diagnosis

Abstract

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 polymerases. 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.

Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast

“…Initial sequencing of the remaining three products in both directions revealed wild-type sequence. Reports in the literature 27,28 indicate that during PCR the wild-type DNA may be amplified preferentially, if wild-type and mutated material are admixed together. Consequently, we cloned these products and, by separating the two alleles, two further mutations were revealed (Table I).…”

Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis

“…However, the effects of heteroduplex formation were not considered in their restriction fragment length polymorphism (RFLP) analyses. 21 Barnard et al 19 found striking differences in the amplification efficiency of wild-type and mutant clones of the p53 and k-ras genes in favor of wild-type sequences, either in a single reaction tube or in different reaction tubes. Although based on a limited number of samples, the data of Bernard et al 19 indicate that a point mutation, not in primer binding sites, can cause PCR bias.…”

“…21 Barnard et al 19 found striking differences in the amplification efficiency of wild-type and mutant clones of the p53 and k-ras genes in favor of wild-type sequences, either in a single reaction tube or in different reaction tubes. Although based on a limited number of samples, the data of Bernard et al 19 indicate that a point mutation, not in primer binding sites, can cause PCR bias. In addition, their data suggest that wild-type sequences derived from normal cells in a clinical sample can potentially mask mutant sequences in a PCR-based assay for mutation detection.…”

“…However, their use of cloned DNA precluded the normalization of template input with a genomic reference sequence for the most accurate quantification of PCR bias. 19 Quantification of PCR bias due to an SNP is complicated by heteroduplex formation between the two sequences, and by the need to quantify nearly identical PCR products independently. We present herein methods to overcome these difficulties.…”

“…Bernard et al 19 hypothesized that the sequence CXGG might cause PCR bias. The segment of SMN1 and SMN2 amplified in our assay contains the sequence .…”