De novo quantitative bisulfite sequencing using the pyrosequencing technology

doi:10.1016/j.ab.2004.05.007

Analytical Biochemistry

Volume 333, Issue 1, 1 October 2004, Pages 119-127

https://doi.org/10.1016/j.ab.2004.05.007 Get rights and content

Abstract

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2–6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.

Section snippets

Materials

Primers for PCR amplification, pyrosequencing, and primer extension reactions were purchased from Biotez (Buch, Germany). Platinum Taq DNA polymerase was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Single-strand DNA-binding protein (SSB), shrimp alkaline phosphatase, and streptavidin Sepharose HP beads were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). TMA31FS DNA polymerase was purchased from Roche Molecular Systems (Alameda, CA, USA). Phosphodiesterase II

Results

To increase the size of the regions that can be analyzed in a single sequencing run, we modified the published protocols [12], [13], [14] on two major points. First, all reactions were run with the SQA reagent kit dedicated to sequencing reactions instead of the SNP reagent kit dedicated to SNP analysis. The SQA reagent kit contains the same components as does the SNP kit (i.e., enzyme and substrate mixtures and nucleotides), but the former is optimized for an increased number of nucleotide

Discussion

DNA methylation analysis has gained growing interest during the past few years in various medical fields, such as development and oncology, due to the increasing number of pathological situations in which it has been found to be involved. However, current methods either allow precise quantification of methylation of several CpGs at once but are labor intensive, precluding analysis of numerous patients, or focus only on one CpG or a few CpGs. Hence, there is an urgent need for a method that

Acknowledgements

This work was supported by the Ministère de l’Education, Recherche, et Technologie (MENRT) of the French government.

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De novo quantitative bisulfite sequencing using the pyrosequencing technology

Abstract

Section snippets

Materials

Results

Discussion

Acknowledgements

J. Biol. Chem.

Curr. Biol.

Curr. Biol.

Trends Genet.

Anal. Biochem.

Anal. Biochem.

DNA methylation in health and disease

Nat. Rev. Genet.

Genomic imprinting: parental influence on the genome

Nat. Rev. Genet.

Epigenomics: genome-wide study of methylation phenomena

Curr. Iss. Mol. Biol.

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands

Proc. Natl. Acad. Sci. USA

DNA methylation: a profile of methods and applications

Biotechniques

The power and the promise of DNA methylation markers

Nat. Rev. Cancer

Denaturing high-performance liquid chromatography: a review

Hum. Mutat.

Pyrosequencing sheds light on DNA sequencing

Genome Res.

Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms

Hum. Mutat.