Optimized Allele-Specific Real-Time PCR Assays for the Detection of Common Mutations in KRAS and BRAF

Abstract

Mutations in the oncogenes KRAS and BRAF have been identified as prognostic factors in patients with colorectal diseases and as predictors of negative outcome in epidermal growth factor receptor-targeted therapies. Therefore, accurate mutation detection in both genes, KRAS and BRAF, is of increasing clinical relevance. We aimed at optimizing allele-specific real-time PCR assays for the detection of common mutations in KRAS and the BRAF Val600Glu mutation using allele-specific PCR primers for allelic discrimination and probes (TaqMan) for quantification. Each reaction mix contains a co-amplified internal control to exclude false-negative results. Allele-specific real-time PCR assays were evaluated on plasmid model systems providing a mutation detection limit of 10 copies of mutant DNA in proportions as low as 1% of the total DNA. Furthermore, we analyzed 125 DNA samples prepared from archived, formalin-fixed, paraffin-embedded colorectal carcinomas and compared results with those obtained from direct-sequence analysis. All mutations determined by sequence analysis could be recovered by allele-specific PCR assays. In addition, allele-specific PCR assays clearly identified three additional samples affected by a mutation. We propose these allele-specific real-time PCR assays as a low-cost and fast diagnostic tool for accurate detection of KRAS and BRAF mutations that can be applied to clinical samples.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine‐needle aspirate samples

“…One group has reported detection of as low as 1% of target DNA using optimized AS-PCR. 23 In cases with higher cellularity and higher percentage of target malignant cells (>20%), there is a perfect concordance between the 2 methods. Our study further demonstrates that when applying a molecular detection system in cytologic specimens with limited cellularity, the method with higher sensitivity prevails with…”

A Simple Two-Stage PCR Method for Quality Amplification of Degenerate DNA

“…This means that the two-stage PCR method reliably amplifies DNA sequences from degenerate template such as FFPE DNA and finds application in HRM analysis and screening of point mutations (manuscripts in preparation). Other PCR-based downstream application where this protocol may be applicable include mutation-specific PCR (allelespecific PCR), methylation-specific PCR and methylation-sensitive HRM [10][11][12]. However, our protocol may not be applicable to the comparative quantitative PCR as differences in the amplification efficiencies between primers for the gene(s) of interest and the housekeeping gene may give spurious gene dosage results over the two amplification stages [13].…”

Current companion diagnostics in advanced colorectal cancer; getting a bigger and better piece of the pie

“…Mutant allele specific PCR uses probes for mutated and non-mutated alleles and allows for enrichment of mutant transcripts in samples with low frequency mutations (35). Probes for each allele are labeled with fluorescent reporter dyes that allow detection and quantification.…”

“…There are a number of modifications to the principle of mutant allele specific PCR that can enhance assay performance, such as peptide-nucleic acid linking or locked nucleic acid incorporation (53). These assays are able to detect mutant allele frequencies that are much lower than Sanger sequencing (as low as 1%) but do not detect mutations outside of those selected for inclusion as primers in the assay (35,36,54). This enhanced sensitivity can result in the re-classification of up to 20% of patients compared to Sanger sequencing or Therascreen and is clinically relevant (30,55).…”