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Sanger Sequencing

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...

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Updated: Jun 27, 2025

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

Modified Unit-Mediated Strand Displacement Reactions for Direct Detection of Single Nucleotide Variants in Active

Hongyan Yu¹, Xiaole Han¹, Weitao Wang¹

¹Key Laboratory of Clinical Laboratory Diagnostics (Chinese Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.

|May 3, 2024

Summary

This study introduces novel methods to enrich active double-stranded DNA (dsDNA) for single nucleotide variant (SNV) detection. These techniques simplify experiments and enable sensitive mutation identification, improving disease diagnosis.

Keywords:

PCR active double-stranded DNA molecular diagnosis single nucleotide variations strand-displacement reactions

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Last Updated: Jun 27, 2025

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Published on: September 25, 2018

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

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Published on: February 13, 2019

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

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Published on: May 6, 2020

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Accurate single nucleotide variant (SNV) identification is crucial for disease diagnosis and treatment.
Current methods often require converting double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) for hybridization, adding complexity and potential information loss.
Fluorescent probes offer potential for rapid and specific SNV detection.

Purpose of the Study:

To develop and evaluate new strategies for enriching active dsDNA for SNV detection.
To investigate the impact of modified units on strand displacement reactions (SDR) and mutation discrimination.
To provide a simpler, faster method for obtaining active dsDNA, facilitating SDR applications.

Main Methods:

Proposed two strategies for active dsDNA enrichment: PCR with obstructive groups and PCR with cleavable units.
Explored the effects of modified units on strand displacement reactions (SDR).
Assessed the discriminatory efficacy of the proposed methods for mutations.

Main Results:

Achieved detection of low variant allele frequencies (VAF) down to 0.1%.
Demonstrated 100% specificity in identifying 45 clinical colorectal cancer tissue samples, with results consistent with sequencing.
The proposed approach offers simple, fast enrichment strategies that preserve original target information.

Conclusions:

The developed strategies effectively enrich active dsDNA, simplifying SNV detection assays.
The methods allow for sensitive and specific identification of mutations, even at low VAFs.
This work facilitates practical applications of SDR based on dsDNA for clinical diagnostics.