ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 333 (2004) 119–127 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.05.007 De novo quantitative bisulWte sequencing using the pyrosequencing technology Jean-Michel Dupont, a,b,¤ Jörg Tost, b Hélène Jammes, c and Ivo Glynne Gut b a Laboratoire d'Histologie Embryologie Cytogénétique, CHU Cochin Port Royal, AP/HP-Université Paris 5, 75014 Paris, France b Centre National de Génotypage, 91057 Evry, France c INSERM U361, CHU Cochin Port Royal, 75014 Paris, France Received 31 March 2004 Available online 24 June 2004 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 quantiWcation of the degree of DNA methylation when 2–6 CpGs were ana- lyzed. 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 bisulWte- treated DNA is necessary to obtain good reproducibility and avoid preferential ampliWcation. 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.  2004 Elsevier Inc. All rights reserved. Keywords: Pyrosequencing; DNA methylation; IGF2; H19; DMR; CpG; BisulWte DNA methylation is a well-recognized epigenetic modiWer in the control of gene expression. This revers- ible DNA modiWcation takes place almost exclusively at cytosine residues associated with guanosine in CpG dou- blets and mediates control of transcription through chromatin conformation changes. It is widely implicated in various biological processes such as the regulation of tissue- and development-speciWc gene expression, X- inactivation, foreign DNA inactivation, and genomic imprinting [1]. In this latter case, the diVerential methyla- tion status of speciWc regulatory regions between the two alleles is responsible for a parent-of-origin speciWc (i.e., either only paternal or only maternal) expression of the imprinted genes. In eVect, there is a loss of heterozygosity of these genes, which consequently behave as though they were haploid [2]. A rough estimate indicates that the human genome contains approximately 200 imprinted genes, of which perhaps 50 are currently known (see cat- alog of imprinted genes and parent-of-origin eVects in humans and animals at http://cancer.otago.ac.nz/igc/ web/home.html). However, very few of them have been well characterized. To fully understand the complex reg- ulation of imprinted regions, one requirement is to pre- cisely localize the diVerentially methylated regions (DMRs) 1 in the vicinity of these genes to elaborate an epigenetic map of the genome [3]. ¤ Corresponding author. Fax: +33-1-58-41-17-55. E-mail address: jean-michel.dupont@cch.ap-hop-paris.fr (J.-M. Dupont). 1 Abbreviations used: DMR, diVerentially methylated region; dH- PLC, denaturing high-performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; SSB, single-strand DNA- binding protein.