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Enhanced Specificity of BRAF V600E Genotyping Using Wild-Type Blocker Coupled with Internal Competitive Reference in
Background:
Mutations in the BRAF gene have been strongly associated with failure in cancer treatment using epidermal growth factor receptor (EGFR) antibodies. To better diagnose and assess the prognosis of cancer patients, mutation screening of the BRAFV600E gene should be performed prior to clinical anti-tumor drug therapy to avoid ineffective treatment.
Methods:
In our previous study, we developed a real-time wild-type blocking PCR (WTB-PCR), which can amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. In order to reduce base mismatch due to the high number of cycles, as well as to monitor the total quantity of DNA added to the reaction system, an internal reference gene was co-amplified together with the target gene on the basis of WTB-PCR.
Results:
Our results showed that when 50 - 200 ng of the DNA templates was used, this current built method (realtime quantitative clamp-based PCR technology using wild-type blocker coupled with internal competitive reference to enhance amplification specificities, named wirePCR) completely blocked the amplification of the wild-type BRAFV600E gene with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000, which was in line with the sensitivity requirement for the detection of trace amounts of the mutant gene. In the colorectal biopsies from 50 patients with suspected colorectal cancer, eight patients (16%) with BRAFV600E mutations were detected using wirePCR. The allele percentage of mutations can be obtained directly from the ΔCq between the targeted and reference genes, we demonstrated that among the V600E-positive patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 24.99% to 54.31%.
Conclusions:
WirePCR is a rapid, simple, and low-cost quantitative analytical technique for the detection of trace amounts of mutant BRAFV600E genes in clinical samples.
Insights
This study introduces wirePCR, a novel method for detecting BRAFV600E mutations. This technique accurately identifies trace amounts of the mutant gene, aiding in personalized cancer treatment decisions.
Area of Science:
- Molecular biology
- Genetics
- Oncology
Background:
- BRAF gene mutations are linked to treatment failure with EGFR antibodies in cancer.
- Accurate BRAFV600E mutation screening is crucial for effective anti-tumor drug therapy and patient prognosis.
Purpose of the Study:
- To develop and validate a sensitive method for detecting BRAFV600E mutations.
- To enable precise quantification of mutant BRAFV600E alleles in clinical samples.
Main Methods:
- Development of real-time quantitative clamp-based PCR technology (wirePCR).
- Utilizes wild-type blocking (WTB-PCR) with an internal reference gene for enhanced specificity and quantification.
- Co-amplification of target and reference genes to monitor DNA quantity and reduce base mismatch.
Main Results:
- WirePCR effectively blocked wild-type BRAFV600E amplification, detecting mutant alleles at a 1:10,000 ratio.
- Identified BRAFV600E mutations in 16% (8/50) of colorectal cancer patients.
- Quantified mutant BRAFV600E allele percentages from 24.99% to 54.31% in positive samples.
Conclusions:
- WirePCR is a rapid, simple, and cost-effective method for detecting trace amounts of mutant BRAFV600E.
- This technique can significantly aid in diagnosing and assessing prognosis for cancer patients.
- Enables avoidance of ineffective treatments by identifying patients with BRAFV600E mutations prior to therapy.