Pyrosequencing™ is an established genetic analysis method based on the principle of sequencing by synthesis. It is the only genetic analysis method capable of

delivering explicit sequence information within minutes. Pyrosequencing is an ideal choice for genetic analysis in clinical research. The output data from

Pyrosequencing is the gold standard of genetic information: real sequence data. This is the best possible assurance of a correct genetic test.

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Chemistry

P YRO S E Q U E N C I N G – O N E H O U R TO E X P L I C I T S E Q U E N C E DATA

PyrosequencingTM for quantitative analysis of CpG methylation

GCA TCpG Methylation Analysis

Among the numerous technologies for methylation analysis, Pyrosequencing represents a breakthrough by combining the processivity of PCR-based technologies with the ability to analyze all the individual CpGs of a given region.

Pyrosequencing gives an unprecedented level of resolution in CpG methylation analysis, faster and easier than by tradi-tional methods.

• High resolution quantification of individual sites

• Built-in QC for bisulfite treatment

• Flexible assays analyse almost any CpG site

• Sensitive to less than 10% methylation

• Results available 30 minutes after PCR

Measure methylation at individual CpG sites

Jean-Michel Dupont, Jörg Tost, Hélène Jammes, and Ivo Glynn Gut, Centre National de Genotypage, Evry, France. (Extracted from “De novo quantitative bisulfite sequencing using the pyrosequencing technology”, Analytical Biochemistry 333 (2004) 119-127).

A G T C A G T C T A G T G A T C A G T C T G A T G A G T C T G

37.5 0.0 35.1 36.6 35.9

Pos 1QC for Bisulfitetreatment

Pos 2 Pos 3 Pos 4High level of reproducibility

Figure 1. High resolution, highly reproducible CpG methylation analysis

Introduction

Pyrosequencing offers a powerful sequence-based analysis method to determine the level of methylation at CpG sites. Exploiting

Pyrosequencing’s unique combination of sequencing and quantification, CpG sites are rapidly analysed to give the individual degree of

methylation, which is then presented in the context of the underlying DNA sequence. Pyrosequencing improves virtually every aspect of

CpG methylation analysis: resolution, speed, accuracy and ease of use, and makes it possible to easily communicate and compare

methylation data. The application of the technology for CpG methylation analysis has been verified by several independent scientific

publications (see references).

Principle of Analysis Using the standard treatment of genomic DNA with

bisulfite (Clark et al, Figure 2), unmethylated Cytosine (C)

is converted to Uracil (U), whereas methylated Cytosine (mC)

remains unchanged. Using PCR, Uracil (U) is amplified to

Thymine (T), whereas methylated Cytosine (mC) is amplified

to Cytosine (C). Discrimination between mC and C is thereby

achieved by transforming mC and C to appear as a C/T SNP

(Figure 3).

Figure 2. Schematic diagram of the bisulfite conversion reaction (from Clark et al, 1994)

Figure 3. An example of a DNA sequence and its conversion by bisulfite treatment and further amplification by PCR. By Pyrosequencing, unmethylated Cytosine, C is measured as the relative content of T at the CpG site, and methylated Cytosine, mC, is measured as the relative content of C at the CpG site.

Preparation of DNA for PyrosequencingThe PCR reaction is performed with one biotinylated PCR primer, which facilitates the conversion of the PCR product to a single-stranded

DNA template suitable for Pyrosequencing. A sequencing primer is added, which anneals to the single stranded DNA template. The time

taken to prepare the DNA for Pyrosequencing is about 15 minutes and can be performed in parallel on 96 samples.

PyrosequencingPyrosequencing can quantify the ratio C/T accurately and reproducibly by sequential addition of the nucleotides (Figure 4). Cytosine is

dispensed to the DNA templates. If incorporation occurs, the intensity of emitted light is proportional to the quantity of incorporated

Cytosine. Then, the same reaction is carried out, this time dispensing Thymine and measuring the intensity of light produced (see Technical

Note 101 “The Principle of Pyrosequencing” for a full description of the detection process).

Figure 4. The Pyrosequencing reaction cascade generates light for every incorporated nucleotide, the intensity of which is proportional to the number of bases incorporated.

Pyrosequencing of the PCR product generates a so-called Pyrogram™, a pictorial representation of sequential nucleotide dispensations plot-

ted against the resultant measured light intensity (Figures 1 and 3). The Pyrogram displays both the nucleotide sequence (as peak sequence)

as well as a quantitative representation of the incorporation events (as peak heights). The degree of methylation is calculated from the peak

heights of C and T:

Workflow of Analysis of CpG methylation by Pyrosequencing

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Assay ResultsResults of a typical CpG methylation analysis are presented in Figure 5.

This particular assay is designed to analyse methylation in Prader-Willi

(PWS) and Angelman (AS) Syndromes, genetic disorders caused by

deletions of an imprinted region on chromosome 15. Different degrees

of methylation distinguish PWS and AS from normal individuals. If the

maternal chromosome region is deleted, methylation is close to 0%,

and if the paternal chromosome region is deleted, methylation is close

to 100%. The orange shaded regions highlight the peaks resulting from

sequential dispensations of C and T from which methylation is

assessed.

Inbuilt Quality ControlsThe data generated by Pyrosequencing contain unique features that act

as quality controls. Firstly, the sequence data gives confirmation that

the analysis was made at the correct sites. Secondly, when the assay

includes analysis of a C not followed by a G, that C should be fully con-

verted to T (T =100 %). This acts as a useful quality control for the full

conversion of unmethylated C to T by the bisulfite treatment and PCR

reaction (blue columns in Figure 5).

ReferencesClark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994; 22(15): 2990-2997.

Explains the principle of bisulfite treatment and PCR amplification of DNA to differentiate between methylated and unmethylated cytosines.

Colella S, Shen L, Baggerly KA, Issa J-PJ, Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. BioTechniques,

Jul 2003; 35: 146-150.

Shows the use of a universal primer for CpG methylation, making the application inexpensive to use.

Dupont JM, Tost J, Jammes H, and Gut IG. De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem, Oct 2004;

333(1): 119-27.

Describes Pyrosequencing as a breakthrough for methylation analysis since it combines the processivity of PCR-based methods with resolving power for

individual quantification of CpG sites.

Tost J, Dunker J, Gut IG. Analysis and quantification of multiple methylation variable positions in CpG islands by PyrosequencingTM. BioTechnigues. Jul

2003; 35: 152-156. Proof statement of Pyrosequencing for CpG methylation, illustrating accuracy and the possibility to analyse multiple CpG sites

Uhlmann K, Brinckmann A, Toliat MR, Ritter H, Nürnberg P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide poly-

morphism analysis. Electrophoresis. 2002; 23: 4072-4079. Pioneering application of Pyrosequencing in CpG methylation analysis.

Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA

elements. Nucleic Acids Res. Feb 2004; 32(3): e38.

Pyrosequencing is applied to the analysis of repetitive DNA elements to assess global methylation content.

Prader Willi/Angelman

Figure 5. Methylation in normal DNA compared to individuals with Prader-Willi and Angelman syndromes

PyrosequencingTM systems are designed for Laboratory Use Only which means that they may be used for either research purposes or by high complexity CLIA certified labs.