sequence of the sense strand, and as adenines in the amplified antisense strand. By incorporating high throughput sequencing adaptors into the PCR primers, PCR products can be sequenced with massively parallel sequencing. Alternatively, and labour-intensively, PCR product can be cloned and sequenced. Nested PCR methods can be used to enhance the product for sequencing. All subsequent DNA methylation analysis techniques using bisulfite-treated DNA is based on this report by Frommer et al. (Figure 2). Although most other modalities are not true sequencing-based techniques, the term "bisulfite sequencing" is often used to describe bisulfite-conversion DNA methylation analysis techniques in general. Pyrosequencing Pyrosequencing has also been used to analyze bisulfite-treated DNA without using methylation-specific PCR. Following PCR amplification of the region of interest, pyrosequencing is used to determine the bisulfite-converted sequence of specific CpG sites in the region. The ratio of C-to-T at individual sites can be determined quantitatively based on the amount of C and T incorporation during the sequence extension. The main limitation of this method is the cost of the technology. However, Pyrosequencing does well allow for extension to high-throughput screening methods. A further improvement to this technique was recently described by Wong et al., which uses allele-specific primers that incorporate single-nucleotide polymorphisms into the sequence of the sequencing primer, thus allowing for separate analysis of maternal and paternal alleles. This technique is of particular usefulness for genomic imprinting analysis. Methylation-sensitive single-strand conformation analysis (MS-SSCA) This method is based on the single-strand conformation polymorphism analysis (SSCA) method developed for single-nucleotide polymorphism (SNP) analysis. SSCA differentiates between single-stranded DNA fragments of identical size but distinct sequence based on differential migration in non-denaturating electrophoresis. In MS-SSCA, this is used to distinguish between bisulfite-treated, PCR-amplified regions containing the CpG sites of interest. Although SSCA lacks sensitivity when only a single nucleotide difference is present, bisulfite treatment frequently makes a number of C-to-T conversions in most regions of interest, and the resulting sensitivity approaches 100%. MS-SSCA also provides semi-quantitative analysis of the degree of DNA methylation based on the ratio of band intensities. However, this method is designed to assess all CpG sites as a whole in the region of interest rather than individual methylation sites. High resolution melting analysis (HRM) A further method to differentiate converted from unconverted bisulfite-treated DNA is using high-resolution melting analysis (HRM), a quantitative PCR-based technique initially designed to distinguish SNPs. The PCR amplicons are analyzed directly by