Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation...

Area of Science:

• Molecular Biology
• Oncology
• Genetics

Background:

• Mutant KRAS proto-oncogene GTPase (KRAS) is crucial for predicting colorectal cancer development, diagnosis, treatment, and targeted therapy efficacy.
• Accurate detection of trace amounts of mutant KRAS is essential for effective patient management.
• Existing methods may face challenges in sensitivity and specificity for low-abundance mutant KRAS detection.

Purpose of the Study:

• To develop and validate a modified locked nucleic acid-based method for improved detection of trace amounts of mutant KRAS.
• To enhance the specificity and sensitivity of mutant KRAS detection in colorectal cancer samples.
• To establish a simple, rapid, and low-cost quantitative analysis method for mutant KRAS detection.

Main Methods:

• Modification of a locked nucleic acid-based method using internal competitive amplification fragments.
• Development of quantitative clamp-based polymerase chain reaction technology with a wild-type blocking (WTB) probe, termed WIRE-PCR.
• Application of WIRE-PCR to colorectal cancer biopsies to detect and quantify mutant KRAS alleles.

Main Results:

• WIRE-PCR effectively blocked wild-type KRAS amplification across various DNA template concentrations (50-150 ng).
• The method demonstrated the ability to detect mutated alleles at a single molecular level.
• Mutant KRAS was detected in 18 out of 50 (36%) colorectal cancer patient biopsies, with mutant DNA levels ranging from 18.6% to 64.2%.

Conclusions:

• WIRE-PCR significantly improves the detection efficacy of trace amounts of mutant KRAS.
• The developed WIRE-PCR method is a simple, rapid, and low-cost tool for quantitative analysis of mutant KRAS.
• This technique holds promise for enhanced diagnosis and monitoring of colorectal cancer patients with KRAS mutations.