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.

Supplemental Figure S1

Amplification curves from KRAS and BRAF PCRs and from respective internal control PCRs. First row: amplification curves of KRAS 12Ala, 12Val, 12Cys, and BRAF 600Glu target-specific real-time PCRs (ie, the fluorescein channel). Second row: amplification curves of respective internal control PCRs (ie, the VIC channel). Ten copies of mutant plasmid DNA were diluted into 990 copies of genomic wild-type DNA. R, reference PCR reaction mix; a, allele-specific PCR reaction mix.

Figure 1

Primer and probes used for KRAS and BRAF real-time PCR. The figure illustrates positions of primer and probes used for KRAS and BRAF real-time PCR. Reference and mutation-specific PCRs share the same probe and the opposite PCR primer. Solid arrows display mutation-unspecific primers; and dotted arrows, mutation-specific primers. Codons affected by the mutation are underlined.

Figure 2

Amplification curves obtained from allele-specific real-time PCR assays. DNA from mutant plasmids was diluted into wild-type plasmids. Based on a total DNA amount of 1000 copies per reaction, the proportion of mutant plasmid DNA was gradually reduced to obtain decreasing ratios of mutant to wild-type DNA. R, reference PCR, representative for all dilutions; a, b, c, and d, 10%, 2.5%, 1%, and 0%, respectively, proportion of mutant DNA; 0, nontemplate control.