As an epigenetic and inheritable trait, DNA methylation is well-known for its role in important biological phenomena, including X-chromosome inactivation (in females), genetic imprinting, cell differentiation, and mutagenesis (Robertson and Wolffe, 2000, Feinberg et al., 2002, Fitzpatrick and Wilson, 2003, Muegge et al., 2003). In the human genome, DNA methylation primarily involves about 70% of all cytosines in the context of 5'-CG-3' (CpG) dinucleotides. Fine mapping of CpG methylation has been a major goal of the Human Epigenome Project (HEP), and the first major product derived from this international effort has been published recently (Rakyan et al., 2004).
Methylated cytosine (mC) differs from cytosine in two biochemical properties. First, treatment of DNA with bisulfite converts normal cytosine to uracil, while mC remains intact. Second, many methylation-sensitive restriction endonucleases are unable to cleave target DNA when mC is present within the recognition site. These properties form the basis for CpG methylation analyses.
Quantification of CpG methylation can be done with a variety of PCR-based techniques (see Table 10.1), each with clear advantages and disadvantages (reviewed by (Geisler et al., 2004)). For example, when a single CpG site is targeted, methylation-specific PCR (MSP) can be used to measure the proportion of mC to normal cytosine in bisulfite-treated DNA samples. However, this technique is ineffective for CpG-rich sequences. For detection and quantification of multiple CpG sites in a single assay, the method of choice seems to be pyrosequencing of PCR-amplified, bisulfite-treated DNA (Tost et al., 2003a, Dupont et al., 2004), while MALDI mass spectrometry represents the latest, high-throughput technology for more demanding projects (Tost et al., 2003b).
This chapter will illustrate the technical aspects of quantitative, real-time PCR (qPCR) in studying de novo CpG methylation. In our analyses of several genes related to the human immunodeficiency virus type 1 (HIV-1) infection and acquired immune deficiency syndrome (AIDS), the method provides a rapid and cost-effective means of identifying samples with differentially methylated CpG sites.
Table 10.1 Common, PCR-based techniques for quantitative analyses of CpG methylation, in which bisulfite-treated DNA samples serve as the template
Key features (advantages and disadvantages)
MSP: methylation-specific PCR
COBRA: combined bisulfite restriction analysis
BiPS: bisulfite PCR-single-strand conformation polymorphism
Simple, sensitive, but only one CpG site at a time
Easy to use, accurate, but not all CpGs are within restriction sites
Rapid, simple, but sensitivity is limited to small fragments
High-throughput, homogenous, and expensive
Sensitive, high-throughput, homogenous, and expensive
Rapid, but only one CpG site at a time
SNuPE: single nucleotide primer extension qPCR: real-time, quantitative PCR Medium- to high-throughput and requires special equipment
Direct sequencing of PCR amplicons
Sequencing of cloned PCR amplicons
Pyrosequencinga of PCR amplicons
MALDIb mass spectrometry
Rapid, accurate, but quantification can be a problem for partially methylated sites
Accurate, time-consuming, and expensive
Rapid, accurate, inexpensive, short reads per assay, and platform is not widely available
Accurate, expensive, and platform is not widely available
(Xiong and Laird, 1997)
(Gonzalgo and Jones, 1997)
Too many to list
aDetailed information can be found at: http://www.pyrosequencing.com/DynPage.aspx?id=7499. bMALDI, matrix-assisted laser desorption/ionization.
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