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This chapter describes a detailed protocol using single-nucleotide primer extension (SNuPE) for quantitative analysis of DNA methylation on specific CpG sites. The first step DNA sample to be studied is treated with sodium bisulfite, which converts selectively unmethylated cytosines to uracil, while methylated cytosines remain unconverted. Subsequently, a SNuPE reaction is performed, with an oligo just flanking a CpG site, using a purified polymerase chain reaction product derived from bisulfite-treated DNA as a template. The oligo is extended by either ddCTP or ddTTP depending on whether the site is methylated or unmethylated, respectively. The reaction is quantitative and linear, and two to three sites can be studied simultaneously in a multiple reaction. The SNuPE product, without further purification, is separated by ion-pair reverse-phase (IP RP) high-performance liquid chromatography (using an alkylated nonporous polysterene-divinylbenzene cartridge) that allows an easy, semiautomated method for separation of the extended and unextended products and an accurate quantification of the extended products. The ratio of the ddCTP to the ddTTP gives the fraction of the methylated cytosines at that specific CpG site. [ABSTRACT FROM AUTHOR]

Genomic imprinting is an epigenetically controlled form of gene regulation leading to the preferential expression of one parental gene copy. To date, approximately 40 imprinted genes have been described that are exclusively or predominantly expressed from either the paternal or the maternal allele (www.mgu.har.mrc.ac.uk/imprinting/implink.html). Changes in the imprinted expression of such genes result in developmental abnormalities; in the human they are associated with several diseases and various types of cancer(1-3). [ABSTRACT FROM AUTHOR]

The postreplicative methylation of DNA at the C5 position of cytosines is found in a broad spectrum of organisms ranging from prokaryotes to human (1). In prokaryotes the major role of cytosine C5 methylation (like adenine N6 and cytosine N4 methylation) is to protect the genome against DNA degrading nucleases (restriction/modification), whereas in many eukaryotes cytosine C5 methylation (found within CpG dinucleotides) plays a pivotal role in the control of gene expression, inactivation of repetitive sequences, stability of chromosomes, and in cell transformation leading to development of cancer. The growing evidence that the cytosine methylation is also crucial in embryonic development of mammals regulating genomic imprinting, X inactivation and cell differentiation (2) has caused a demand for effective methods that would detect this modification with high sensitivity and reliability. [ABSTRACT FROM AUTHOR]