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Updated: Jun 14, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
KRas and BRaf mutational status analysis from formalin-fixed, paraffin-embedded tissues using multiplex polymerase
Arturas Jakubauskas1, Laimonas Griskevicius
1Department of Molecular and Regenerative Medicine, Oncology and Transfusion Medicine Center, Vilnius University Hospital, Santariskiu Clinics, Lithuania. arturas.jakubauskas@santa.lt
Context:
Monoclonal anti-epidermal growth factor receptor antibodies bind to the epidermal growth factor receptor and inhibit receptor kinase activity. Clinical trials have indicated that evaluation of the mutational status of KRas and BRaf is necessary to exclude patients who are resistant to the clinical benefit of anti-epidermal growth factor receptor therapy.
Objective:
To develop a multiplex polymerase chain reaction-based assay for the evaluation of KRas and BRaf mutational status.
Design:
A sample-saving and cost-effective, multiplex polymerase chain reaction-based assay to detect somatic mutations in KRAS exon 2 and exon 3 as well as in BRAF exon 15 was developed. The same primer pairs could be successfully used in amplification of a single DNA fragment under the same conditions.
Results:
DNA isolated from 20 retrospective formalin-fixed, paraffin-embedded samples of colorectal cancer was screened for mutations using the multiplex polymerase chain reaction assay followed by dideoxy-termination sequencing. Five samples bearing mutations-p.G12D (identified twice), p.G12V, p.G12S, and p.G13D, all encoded in KRAS exon 2-were identified. Three samples were found bearing amino acid substitution p.V600E of BRaf. The detected KRas and BRaf mutations were found to be mutually exclusive.
Conclusions:
A multiplex polymerase chain reaction-based amplification followed by dideoxy-termination sequencing may be used advantageously for the evaluation of KRas and BRaf mutational status from formalin-fixed, paraffin-embedded samples.
Insights
A new multiplex PCR assay efficiently detects KRas and BRaf mutations in colorectal cancer. This method aids in identifying patients resistant to anti-EGFR therapy, improving treatment selection.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Monoclonal anti-epidermal growth factor receptor (EGFR) antibodies inhibit EGFR kinase activity.
- KRas and BRaf mutational status is crucial for predicting response to anti-EGFR therapy.
- Identifying patients resistant to therapy is essential for effective treatment strategies.
Purpose of the Study:
- To develop a multiplex polymerase chain reaction (PCR)-based assay.
- To evaluate KRas and BRaf mutational status for clinical application.
- To provide a sample-saving and cost-effective diagnostic tool.
Main Methods:
- Developed a multiplex PCR assay for KRAS exon 2/3 and BRAF exon 15 mutations.
- Utilized a single set of primer pairs for efficient DNA fragment amplification.
- Screened 20 formalin-fixed, paraffin-embedded colorectal cancer samples.
Main Results:
- Identified KRAS mutations (p.G12D, p.G12V, p.G12S, p.G13D) in 5 samples.
- Detected BRAF V600E mutation in 3 samples.
- Observed that KRas and BRaf mutations were mutually exclusive.
Conclusions:
- Multiplex PCR followed by dideoxy-termination sequencing is effective for KRas/BRaf mutation analysis.
- This assay is advantageous for evaluating mutational status in FFPE samples.
- The method supports personalized medicine by guiding anti-EGFR therapy selection.
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