ABSTRACT
I. Introduction 140
II. Materials 140
A. Bisulfite Treatment 140
B. Purification of Bisulfite-Treated PCR Product 140
C. SNuPE Reaction and HPLC Analysis 140
III. Bisulfite Treatment and Amplification 141
A. Principle of the Bisulfite Conversion Reaction 141
B. Protocol for Treatment of Intact Cells 141
C. Protocol for Treatment of Isolated DNA 142
D. Selective Amplification of Converted DNA 142
1. Primer Design 142
2. Optimizing PCR Conditions 143
E. Troubleshooting 143
F. Drawbacks of the Method 144
IV. Quantification 144 A. Cloning and Sequencing 144
B. Analysis by SIRPH (SNuPE-IP RP HPLC) 144 1. Protocol Outline 145
2. Guidelines for Designing SNuPE Primers 146 3. Analysis of the Results 146 4. Troubleshooting 147
5. Advantages and Drawbacks of the SIRPH Method 147 V. Conclusions 147
Acknowledgments 147
References 147
I. INTRODUCTION
The most advanced and detailed method to analyze cytosine methylation in genomic DNA is the bisulfite-based sequencing introduced by Frommer and colleagues in 1992.1 It is based on bisulfite treatment which converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged under the experimental conditions used. The great potential of this method was quickly
recognized, and a number of improvements of the original protocol were published, mostly to achieve better sensitivity and conversion rates (a systematic analysis of reaction parameters is given in Jost et al.).2 Other improvements focused on use of the method on imbedded tissues and intact cells. In 1996, we published a protocol in which the whole reaction is performed on material — DNA or cells-embedded in low-melting-point (LMP) agarose.3 This approach proved to be superior to other protocols in several aspects. It facilitates sample handling by eliminating precipitation steps and does not require DNA isolation out of cells or early embryos. It thereby reduces loss of material and allows the assessment of minute sample quantities. Furthermore, the physical trapping of the DNA within the agarose matrix prevents renaturation and thus helps to keep the DNA single-stranded, an indispensable prerequisite for an efficient conversion reaction. After generating a PCR product, the next step in a bisulfite analysis is to quantitate the ratio of cytosine to thymine content at a specific CpG site. To address this, many methods have been developed. In this chapter, we will describe in detail the traditional cloning and sequencing method (Section IV.A) and compare this with our recently published SIRPH protocol4 (Section IV.B).
