Analysing DNA methylation using bisulphite pyrosequencing.
作者： Thomas Mikeska , Jörg Felsberg , Chelsee A. Hewitt , Alexander Dobrovic
DOI: 10.1007/978-1-61779-316-5_4
关键词: DNA methylation 、 Illumina Methylation Assay 、 Computational biology 、 CpG site 、 Chemistry 、 Pyrosequencing 、 Primer (molecular biology) 、 Methylation 、 Amplicon 、 DNA sequencing
摘要: Bisulphite pyrosequencing is a quantitative methodology for the investigation of DNA methylation sequences up to 100-bp in length. Biotin-labelled, single-stranded PCR products generated from bisulphite-treated are used as template with an internal primer perform reaction. Nucleotides added predetermined order each cycle and amount incorporated nucleotide results proportional emission light. ratios calculated levels light emitted at individual CpG positions strand-dependent manner. The detection limit sites approximately 5% displayed average level position assayed across all amplification during As consequence, bisulphite allows identification heterogeneous patterns but does not provide information single allele resolution. This suited analyse short such those typically extracted formalin-fixed paraffin-embedded specimens. Nevertheless, longer can be sequenced by serial pyrosequencing, which utilises tandem assays along amplicon. general provided applicable formats current instruments, however, specific protocol PyroMark Q24 instrument provided.
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