Related Experiment Video
Updated: Sep 21, 2025
Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues
Published on: August 23, 2024
An ultra-sensitive method to detect mutations in human RAS templates
Siqi Li1,2, Christopher M Counter1
1Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, NC, USA.
Abstract:
The RAS family of small GTPases is mutated in roughly a fifth of human cancers. Hotspot point mutations at codons G12, G13, and Q61 account for 95% of all these mutations, which are well established to render the encoded proteins oncogenic. In humans, this family comprises three genes: HRAS, NRAS, and KRAS. Accumulating evidence argues that oncogenic RAS point mutations may be initiating, as they are often truncal in human tumours and capable of inducing tumorigenesis in mice. As such, there is great interest in detecting oncogenic mutation in the RAS genes to understand the origins of cancer, as well as for early detection purposes. To this end, we previously adapted the microbial ultra-sensitive aximum epth equencing (MDS) assay for the murine Kras gene, which was capable of detecting oncogenic mutations in the tissues of mice days after carcinogen exposure, essentially capturing the very first step in tumour initiation. Given this, we report here the adaption and details of this assay to detect mutations in a human KRAS sequence at an analytic sensitivity of one mutation in a million independently barcoded templates. This humanized version of MDS can thus be exploited to detect oncogenic mutations in KRAS at an incredible sensitivity and modified for the same purpose for the other RAS genes.
Insights
We developed a highly sensitive assay to detect cancer-driving mutations in the KRAS gene. This method can identify rare oncogenic mutations, aiding in early cancer detection and understanding tumor origins.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- RAS family small GTPases are frequently mutated in human cancers, with specific hotspot mutations driving oncogenesis.
- Oncogenic RAS mutations are often early events in tumorigenesis, making their detection crucial for understanding cancer origins and early detection.
- Previous work adapted a Maximum Depth Sequencing (MDS) assay for murine Kras, demonstrating its ability to detect mutations shortly after carcinogen exposure.
Purpose of the Study:
- To adapt and detail an ultra-sensitive Maximum Depth Sequencing (MDS) assay for detecting mutations in the human KRAS gene.
- To achieve a high analytic sensitivity for detecting oncogenic KRAS mutations, enabling the identification of rare mutational events.
- To establish a foundation for applying this sensitive assay to other RAS family genes for comprehensive cancer mutation analysis.
Main Methods:
- Adaptation of the microbial ultra-sensitive Maximum Depth Sequencing (MDS) assay for a human KRAS sequence.
- Optimization of the assay to achieve an analytic sensitivity of one mutation in a million independently barcoded templates.
- Validation of the assay's capability to detect oncogenic mutations in human KRAS.
Main Results:
- The humanized MDS assay successfully detects mutations in a human KRAS sequence.
- The assay demonstrates an analytic sensitivity of one in a million independently barcoded templates for KRAS mutations.
- This high sensitivity allows for the detection of rare oncogenic mutations, crucial for early cancer detection.
Conclusions:
- The adapted MDS assay provides an ultra-sensitive method for detecting oncogenic mutations in human KRAS.
- This technology can be valuable for early cancer detection and for studying the initiating events of RAS-driven cancers.
- The assay is adaptable for detecting mutations in other RAS family genes, offering a versatile tool for cancer research.
More Related Videos
10:41Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
Published on: March 29, 2017
10:16Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
