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Updated: Jun 30, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Published on: January 8, 2008

De novo quantitative bisulfite sequencing using the pyrosequencing technology.

Jean-Michel Dupont¹, Jörg Tost, Hélène Jammes

¹Laboratoire d'Histologie Embryologie Cytogénétique, CHU Cochin Port Royal, AP/HP-Université Paris 5, 75014 Paris, France. jean-michel.dupont@cch.ap-hop-paris.fr

Analytical Biochemistry

|September 8, 2004

Summary

This study enhances DNA methylation analysis using pyrosequencing, enabling the accurate quantification of up to 10 CpG sites in a single run. This improved epigenotype-mapping tool offers a more efficient method for studying DNA methylation patterns.

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Last Updated: Jun 30, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping

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Published on: January 8, 2008

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing for Microbial Identification and Characterization

Published on: August 22, 2013

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Published on: February 24, 2015

Area of Science:

Molecular Biology
Genetics
Epigenetics

Background:

Current DNA methylation analysis methods are often labor-intensive or limited in scope.
Pyrosequencing offers a potential solution for more comprehensive DNA methylation analysis.

Purpose of the Study:

To improve pyrosequencing protocols for analyzing a larger number of CpG sites in a single reaction.
To establish a robust and reproducible method for quantitative DNA methylation analysis.

Main Methods:

Optimization of pyrosequencing enzyme mix and inclusion of single-stranded DNA-binding protein.
Analysis of up to 10 successive CpGs within a 75-nucleotide region.
Application to CpG islands near the IGF2 and H19 genes using bisulfite-treated DNA.

Main Results:

Reproducible quantification of DNA methylation for up to 10 contiguous CpGs was achieved.
A minimum of 10 ng of bisulfite-treated DNA was identified as necessary for optimal results.
Accurate, quantitative de novo sequencing of methylation states revealed reproducible variations in contiguous CpGs.

Conclusions:

The enhanced pyrosequencing protocol significantly improves the capacity for DNA methylation analysis.
This method provides a valuable tool for studying complex DNA methylation patterns, complementing existing technologies.