分子诊断原理与技术.pptx

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,#,分子诊断原理与技术,分子诊断原理与技术,第1页,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.,分子诊断原理与技术,第2页,分子诊断原理与技术,第3页,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 CphosphateG,that is,cytosine and guanine separated by a,phosphate,which links the two,nucleosides,together in DNA.,CpGnotation 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%.,分子诊断原理与技术,第4页,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.,分子诊断原理与技术,第5页,CpG,Islands,(CGI),search Online Resource,cpgislands.usc.edu/,www.ebi.ac.uk/Tools/emboss/cpgplot/index.html,分子诊断原理与技术,第6页,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,分子诊断原理与技术,第7页,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,分子诊断原理与技术,第8页,分子诊断原理与技术,第9页,分子诊断原理与技术,第10页,Direct sequencing,The first reported method of methylation analysis using bisulfite-treated 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 bisulfite-specific(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.,分子诊断原理与技术,第11页,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.,Direct Sequencing,分子诊断原理与技术,第12页,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,Pyrosequencing does well allow for extension to,high-throughput screening,methods.,分子诊断原理与技术,第13页,FLASH,分子诊断原理与技术,第14页,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.,分子诊断原理与技术,第15页,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.,The MS-HRM assay for,BNIP3,methylation.Results of the,BNIP3,-MS-HRM assay for five clinical samples compared to the dilution standards.,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,.,分子诊断原理与技术,第16页,Methylation-sensitive single nucleotide primer extension(MS-SnuPE),分子诊断原理与技术,第17页,Analysis of methylation by base-specific cleavage and MALDI-TOF MS,Ehrich M et al.PNAS;102:15785-15790,by National Academy of Sciences,分子诊断原理与技术,第18页,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 bisulfite-converted genomic DNA.Such primers will only anneal to sequences that are methylated,and thus containing,5-methylcytosines,that are resistant to conversion by bisulfite.Alternatively,unmethylated-specific primers can be used.,分子诊断原理与技术,第19页,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.,分子诊断原理与技术,第20页,Nucleic Acids Research 34(11):e82,分子诊断原理与技术,第21页,Copyright restrictions may apply.,Gebhard,C.et al.Nucl.Acids Res.34:e82;doi:10.1093/nar/gkl437,Outline of MB-PCR,分子诊断原理与技术,第22页,Copyright restrictions may apply.,Gebhard,C.et al.Nucl.Acids Res.34:e82;doi:10.1093/nar/gkl437,MB-PCR detects methylation of CpG island promoters,分子诊断原理与技术,第23页,Copyright restrictions may apply.,Gebhard,C.et al.Nucl.Acids Res.34:e82;doi:10.1093/nar/gkl437,Detecting CpG methylation in leukemia cell lines by MB-PCR,分子诊断原理与技术,第24页,Copyright restrictions may apply.,Gebhard,C.et al.Nucl.Acids Res.34:e82;doi:10.1093/nar/gkl437,Methylation of the ICSBP promoter inversely correlates with ICSBP expression in leukemia cell lines,分子诊断原理与技术,第25页,Copyright restrictions may apply.,Gebhard,C.et al.Nucl.Acids Res.34:e82;doi:10.1093/nar/gkl437,Sensitivity of MB-PCR,分子诊断原理与技术,第26页,Copyright restrictions may apply.,Gebhard,C.et al.Nucl.Acids Res.34:e82;doi:10.1093/nar/gkl437,Detection of aberrant CpG methylation in primary AML blasts,分子诊断原理与技术,第27页,Fragile X syndrome,脆性,X,综合征,Location of,FMR1,gene,intellectual disability,elongated face,large ears,flat feet,larger testes,low muscle tone,cluttered speech,nervous speech,分子诊断原理与技术,第28页,FMR1,(,fragile X mental retardation 1,),is a,human,gene,that codes for a,protein,called,f,ragile X,m,ental,r,etardation,p,rotein,or FMRP.,This protein is normally made in many tissues,especially in the,brain,and,testes,.It may play a role in the development of,synaptic,connections between,nerve cells,in the brain,where cell-to-cell communication occurs.The connections between nerve cells can change and adapt over time in response to experience(a characteristic called,synaptic plasticity,).,FMRP may help regulate synaptic plasticity,which is important for learning and memory.,FMR1 has been shown to interact with FXR2,CYFIP1,CYFIP2,NUFIP1,FXR1 and NUFIP2.,分子诊断原理与技术,第29页,分子诊断原理与技术,第30页,分子诊断原理与技术,第31页,Expression of the,FMR1,Gene and Associated Clinical Disorders,分子诊断原理与技术,第32页,The fragile X region with normal,premutation,and full mutation CGG repeats.The CGG repeat region is represented by a jagged line.Restriction sites are indicated by solid arrows for,Eco,RI and a dashed arrow for methylation sensitiveEag,I.Full mutation alleles are typically methylated and are resistant to digestion at the,Eag,I site.,分子诊断原理与技术,第33页,Restriction map of the fragile X region.Probes used in Southern analysis of the region are shown.,分子诊断原理与技术,第34页,Fragile X Southern analysis of genomic DNA digested with,Eco,RI and,Eag,I,and hybridized with probe StB12.3.Lane 1:full mutation male with methylation mosaicism.Lane 2:premutation male.Lane 3:premutation female.Lane 4:full mutation male.Lane 5:full mutation female.Lane 6:Full mutation male.Lanes 7 to 9:normal females.Lane 10:full mutation male.DNA size markers are indicated on the right.,分子诊断原理与技术,第35页,