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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...

Next-generation Sequencing

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....

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Updated: Jun 17, 2025

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

Highly accurate single-color fluorogenic DNA decoding sequencing for mutational genotyping.

Chu Cheng¹, Qingzhou Cheng¹, Wei Zhou¹

¹College of Medicine and Health Science, Wuhan Polytechnic University, Wuhan, China.

Journal of Pharmaceutical and Biomedical Analysis

|August 7, 2024

Summary

This summary is machine-generated.

A novel single-color DNA sequencing method enhances accuracy and read length by using modified nucleotides. This approach significantly reduces sequencing errors, offering a more cost-effective alternative for biological and medical applications.

Keywords:

Decoding sequencing High accuracy Long read length Rare mutations Single-color fluorogenic DNA

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Current DNA sequencing technologies face limitations in accuracy, read length, and throughput.
Reducing sequencing costs and error rates is crucial for broader applications in research and clinical settings.

Purpose of the Study:

To develop a single-color fluorogenic DNA decoding sequencing method.
To improve sequencing accuracy, read length, throughput, and reduce scanning time.
To enable efficient detection and correction of sequencing errors and mutation sites.

Main Methods:

Incorporation of four types of 3'-O-modified nucleotide reversible terminators, with two labeled by the same fluorophore and two unlabeled.
Cyclic interrogation of DNA templates twice with different nucleotide combinations to obtain sequential encodings.
Demonstration of feasibility using established sequencing chemistry and validation on human mitochondrial DNA.

Main Results:

Achieved a cycle efficiency of approximately 99.5%.
Demonstrated a theoretical error rate of 0.00016% at a sequencing depth of ×2, outperforming Sanger sequencing.
Successfully detected a mutation site in human mitochondrial DNA from a single sequencing run.

Conclusions:

The proposed single-color fluorogenic DNA sequencing method offers significant improvements in accuracy, read length, and efficiency.
The method is compatible with existing sequencing-by-synthesis platforms and has the potential to reduce costs.
This approach holds promise for advancing applications in various fields of biology and medicine.