分子诊断原理与技术(一).pptVIP

* DNA methylation Concept Detection Application DNA methylation involves the addition of a methyl group to the 5 position of cytosine, which occurs in the context of CpG (cytosine followed by guanine) dinucleotides. This modification can be inherited through cell division. CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. CpG is shorthand for —C—phosphate—G—, that is, cytosine and guanine separated by a phosphate, which links the two nucleosides together in DNA. CpG notation is used to distinguish this linear sequence from the base-pairing of cytosine and guanine. The frequency of CpG dinucleotides in human genomes is 1% . There are regions of the DNA that have a higher concentration of CpG sites, known as CpG islands. Many genes in mammalian genomes have CpG islands associated with the start of the gene. Because of this, the presence of a CpG island is used to help in the prediction and annotation of genes. CpG Islands (CGI) search Online Resource / http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html Methods 1 Non-methylation-specific PCR based methods Direct sequencing Pyrosequencing Methylation-sensitive single-strand conformation analysis (MS-SSCA) High resolution melting analysis (HRM) Methylation-sensitive single nucleotide primer extension (MS-SnuPE) Base-specific cleavage/MALDI-TOF 2 Methylation-specific PCR (MSP) 3 Microarray-based methods Treatment of DNA with bisulfite* converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. *亚硫酸氢盐 Direct sequencing The first reported method of methylation analysis using bisulfite-treated DNA utilized PCR and stand