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Updated: Apr 22, 2026

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Published on: March 29, 2017

Detecting ultralow-frequency mutations by Duplex Sequencing.

Scott R Kennedy¹, Michael W Schmitt², Edward J Fox¹

¹Department of Pathology, University of Washington, Seattle, USA.

Nature Protocols

|October 10, 2014

Summary

This summary is machine-generated.

Duplex Sequencing (DS) detects rare DNA mutations with high accuracy. This method analyzes both strands of DNA to identify single nucleotide changes in large populations, ideal for studying genetic alterations.

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Last Updated: Apr 22, 2026

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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Published on: September 25, 2018

Area of Science:

Genomics
Molecular Biology
Biotechnology

Background:

Next-generation sequencing enables detection of genetic alterations.
Studying heterogeneous populations requires sensitive mutation detection methods.
Existing methods may lack the sensitivity to detect very-low-frequency genetic alterations.

Purpose of the Study:

To provide a detailed protocol for Duplex Sequencing (DS).
To enable the study of heterogeneous populations and very-low-frequency genetic alterations.
To optimize DS for small genomic regions (<1 Mb).

Main Methods:

Duplex Sequencing (DS) utilizes adapter ligation to sample DNA.
Sequencing adapters contain complementary double-stranded nucleotide sequences.
PCR amplification creates sequence families from original DNA strands.
Mutations are confirmed if present on PCR families from both DNA strands.

Main Results:

DS achieves high sensitivity, detecting single mutations among >1 × 10(7) wild-type nucleotides.
The protocol covers adapter synthesis, library preparation, and target enrichment.
The data analysis workflow is outlined.
The protocol can be completed in 1-3 days.

Conclusions:

Duplex Sequencing is a powerful tool for detecting low-frequency DNA mutations.
The provided protocol facilitates the application of DS in research.
DS is suitable for analyzing heterogeneous DNA samples and small genomic regions.