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methylSEQr™ System: Bisulfite Conversion and Sequencing of Methylated DNA
Sponsored,vendor-submitted protocol   Published in December 2006 2007 (p.33) Abstract

DNA methylation patterns—are often considered as a second code, an additional layer of information superimposed on the DNA code that determines many phenotypic attributes.

DNA methylation patterns—are often considered as a second code, an additional layer of information superimposed on the DNA code that determines many phenotypic attributes. The DNA code is stable, but DNA methylation patterns change in response to spatial, temporal and environmental cues. After PCR amplification, the sequence of the bisulfite converted DNA will have C residues only if the C was methylated and will occur adjacent to G's (CpG). All other non-methylated C's will be detected as T's. An unmethylated gDNA sample will have no C's in the sequencing data. Comparison of sodium bisulfite treated DNA sequences with sequences obtained from untreated genomic DNA allows the precise identification of all methylated cytosines within a stretch of DNA.

PCR amplicons generated after bisulfite conversion can be cloned and sequenced or sequenced directly.

The methylSEQr™ Bisulfite Conversion Kit uses a straightforward procedure based on conveniently packaged bisulfite-conversion re-agents and an efficient purification scheme which reliably provides a high recovery of the bisulfite-converted gDNA. Bisulfite treatment converts non-methylated cytosine (C) to uracil (U). Methylated C residues are protected from the conversion. Non-methylated C residues are detected by the C to T transition in the treated DNA sequence. DNA methylation is a common biochemical modification of eukaryotic DNA. Selective gene inactivation has been shown to be due to the DNA methylation of cytosines in the promoter regions.

Protocol Summary

The protocol for methylation sequencing is divided into three main stages:

Sample Preparation and bisulfite conversion

PCR template Prep

Sequencing



methylSEQr™ Bisulfite Conversion Kit Protocol:

  1. Genomic DNA

    1. Prepare genomic DNA according to standard protocols.

  2. Bisulfite Conversion

    1. To gDNA, add MethylSEQr™ Denaturation Buffer. Mix and incubate.

    2. Add MethylSEQr™ Conversion Reagent. Mix and incubate.

  3. Cleanup

    1. Centrifuge the mixture through the methylSEQr™ column.

    2. Water washes.

    3. Add TE and mix. Incubate 5 min.

  4. Template Prep

    1. Primer Design: Applied Biosystems offers a free design tool, Methyl Primer Express® Software for the PCR primer design of bisulfite sequencing and for methylation-specific PCR (MSP). Methyl Primer Express® software can be downloaded from Web site (external)

    2. PCR reaction: We recommend using forward and reverse primers that have been tailed with -21 M13.

    3. PCR cleanup: Add ExoSAP-IT® (USB Corporation), incubate and heat.

  5. Sequencing

    1. Sequencing Reaction: For 10 µl sequencing reactions – BigDye® Terminator Ready ReactionMix v3.1; M13 forward or reverse primer; PCR product.

    2. Sequencing Reaction Cleanup

    3. Data Analysis: Sequencing is challenging due to the decrease in cytosine or guaninenucleotides, therefore we recommend performing electrophoresis with the Applied Biosystems 3130xl Genetic Analyzer or the Applied Biosystems 3730xl DNA Analyzer and the KB™ Basecaller Software to avoid mis-calling. Detection of C to T conversion can be automated with SeqScape® software.

For more information – References

MethlySEQr™ Kit Protocol Summary – PN107PT01-01 Applied Biosystems

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