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Updated: Jun 9, 2026
Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
Published on: August 23, 2024
High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type
Qing Huang1, Gui-Yu Wang, Jun-Fu Huang
1Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
Abstract:
The missense mutations at codons 12 and 13 of KRAS gene have been confirmed as a predictor of nonresponse to EGFR-targeted therapy with monoclonal antibodies cetuximab and panitumumab in patients with metastatic colorectal carcinoma (mCRC). Because of the intra-tumor heterogeneity at genetic levels, it is important to develop sensitive and selective assays to detect above KRAS mutation of rare mutated cells in the presence of large excess of wild-type cells. In the present study, wild-type blocking PCR (WTB-PCR) was developed to detect the aforementioned KRAS mutations, in which a chimera composed of locked nucleic acid (LNA) and DNA was used to inhibit with high sensitivity the amplification of wild-type KRAS alleles whereas it allowed the highly selective amplification of mutated KRAS alleles. Using mutated KRAS from HCT-116 as spiking DNA, the results showed that WTB-PCR could detect mutated alleles in a ratio of 1:10,000 (i.e., 0.01%) wild-type alleles and at a single copy level. For its further applications to detect aforementioned KRAS mutations in 20 cases of mCRC patients, the results showed that the detected mutation percentage of WTB-PCR (60%, 12/20) was higher than that of traditional PCR (45%, 9/20). Moreover, two patients respectively having synonymous mutated codons 13 (i.e., c.39C > T) and missense mutated codons 14 (i.e., c.40G > A) could be also only detected by WTB-PCR. In conclusion, the current WTB-PCR was a rapid, simple, and low-cost method to detect a trace amount of mutated KRAS gene.
Insights
A new Wild-Type Blocking PCR (WTB-PCR) method accurately detects rare KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. This sensitive assay improves detection rates compared to traditional PCR, aiding treatment decisions.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- KRAS gene mutations (codons 12 and 13) predict non-response to EGFR-targeted therapies like cetuximab and panitumumab in metastatic colorectal carcinoma (mCRC).
- Intra-tumor heterogeneity necessitates sensitive assays for detecting rare mutated KRAS alleles amidst abundant wild-type alleles.
Purpose of the Study:
- To develop a highly sensitive and selective assay for detecting KRAS mutations (codons 12 and 13) in mCRC patients.
- To evaluate the efficacy of the developed Wild-Type Blocking PCR (WTB-PCR) method in comparison to traditional PCR.
Main Methods:
- Development of Wild-Type Blocking PCR (WTB-PCR) utilizing a locked nucleic acid (LNA)-DNA chimera to inhibit wild-type KRAS amplification.
- Spiking experiments with mutated KRAS DNA demonstrated WTB-PCR's ability to detect mutated alleles at a 1:10,000 ratio (0.01%) and at a single-copy level.
- Application of WTB-PCR to 20 mCRC patient samples for detecting KRAS mutations.
Main Results:
- WTB-PCR detected KRAS mutations in 60% (12/20) of mCRC patients, outperforming traditional PCR at 45% (9/20).
- The assay successfully identified rare mutations, including synonymous mutations at codon 13 (c.39C>T) and missense mutations at codon 14 (c.40G>A) in two patients.
- WTB-PCR demonstrated high sensitivity, detecting mutated alleles down to 0.01% and single-copy levels.
Conclusions:
- The developed WTB-PCR is a rapid, simple, and cost-effective method for detecting trace amounts of mutated KRAS.
- This assay offers improved detection sensitivity for KRAS mutations in mCRC, potentially impacting treatment strategies.
- WTB-PCR's ability to detect rare and specific mutations enhances its clinical utility in managing mCRC.
