分子诊断原理与技术(一)

分子诊断 生物与医学类研究生课程

DNA methylationConcept Detection Application

分子诊断 生物与医学类研究生课程

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.

分子诊断 生物与医学类研究生课程

分子诊断 生物与医学类研究生课程

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% .

分子诊断 生物与医学类研究生课程

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.

分子诊断 生物与医学类研究生课程

CpG Islands (CGI) search Online Resourcec.edu/ http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html

分子诊断 生物与医学类研究生课程

Methods 1 Non-methylation-specific PCR based methodsDirect 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

分子诊断 生物与医学类研究生课程

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 亚硫酸氢盐

分子诊断 生物与医学类研究生课程

分子诊断 生物与医学类研究生课程

分子诊断 生物与医学类研究生课程

Direct sequencingThe first reported method of methylation analysis using bisulfitetreated DNA utilized PCR and standard dideoxynucleotide DNA sequencing to directly determine the nucleotides resistant to bisulfite conversion. Primers are designed to be strand-specific as well as bisulfitespecific (i.e., primers containing non-CpG cytosines such that they are not complementary to non-bisulfite-treated DNA), flanking (but not involving) the methylation site of interest.

分子诊断 生物与医学类研究生课程

Direct Sequencing This technique required cloning of the PCR product prior to sequencing for adequate sensitivity, and therefore was a very labour-intensive method unsuitable for higher throughput.

分子诊断 生物与医学类研究生课程

Pyrosequencing 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, Pyrosequen

cing does well allow for extension to high-throughput screening methods.

分子诊断 生物与医学类研究生课程

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分子诊断 生物与医学类研究生课程

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.

This method is ideally designed to assess all CpG sites as a whole in the region of interest rather than individual methylation sites.

分子诊断 生物与医学类研究生课程

High resolution melting analysis (HRM) A further method to differentiate converted from unconverted bisulfite-treated DNA is using high resolution melting analysis (HRM), a real-time PCR-based technique initially designed to distinguish SNPs.

This method allows direct quantitation in a single-tube assay, but again assesses methylation in the amplified region as a whole rather than at specific CpG sites.

The MS-HRM assay for BNIP3 methylation. Results of the BNIP3-MSHRM assay for five clinical samples compared to the dilution standards.

分子诊断 生物与医学类研究生课程

Methylation-sensitive single nucleotide primer extension (MS-SnuPE)

分子诊断 生物与医学类研究生课程

Analysis of methylation by base-specific cleavage and MALDI-TOF MS

Ehrich M et al. PNAS 2005;102:15785-15790

©2005 by National Academy of Sciences

分子诊断 生物与医学类研究生课程

Methylation-specific PCR (MSP)

Methylation-specific PCR is a sensitive method to discriminately amplify and detect a methylated region of interest using methylated-specific primers on bisulfiteconverted genomic DNA. Such primers will only anneal to sequences that are methylated, and thus containing 5methylcytosines that are resistant to conversion by bisulfite. Alternatively, unmethylated-specific primers can be used.

分子诊断 生物与医学类研究生课程

Microarray-based methods Microarray-based methods are a logical extension of the technologies available to analyze bisulfite-treated DNA to allow for genome-wide analysis of methylation.

分子诊断 生物与医学类研究生课程

Nucleic Acids Research 2006 34(11):e82

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