tools of molecular genetics-final20141217

1、The application of tools of Human Molecular GeneticsDan wu Peking University Health Science Center Department of Medical 分支学科分支学科 1细胞遗传学细胞遗传学(cytogenetics) 2生化遗传学生化遗传学(biochemical genetics) 3分子遗传学分子遗传学(molecular genetics) 4群体遗传学群体遗传学(population genetics) 5药物遗传学药物遗传学(pharmacogenetics) 6免疫遗传学免疫遗传学(imm

2、uogenetics) 7肿瘤遗传学肿瘤遗传学(cancer genetics) 8毒理遗传学毒理遗传学(toxicology genetics) 9. 发育遗传学发育遗传学(developmental genetics) 10. 行为遗传学行为遗传学(behavior genetics) 11. 基因工程基因工程(genetic engineering) 12. 优生学优生学(eugenics)分支学科分支学科 1细胞遗传学细胞遗传学(cytogenetics) 2生化遗传学生化遗传学(biochemical genetics) 3分子遗传学分子遗传学(molecular genetics)

3、 4群体遗传学群体遗传学(population genetics) 5药物遗传学药物遗传学(pharmacogenetics) 6免疫遗传学免疫遗传学(immuogenetics) 7肿瘤遗传学肿瘤遗传学(cancer genetics) 8毒理遗传学毒理遗传学(toxicology genetics) 9. 发育遗传学发育遗传学(developmental genetics) 10. 行为遗传学行为遗传学(behavior genetics) 11. 基因工程基因工程(genetic engineering) 12. 优生学优生学(eugenics)Analysis of individua

4、l DNA and RNA sequenceslMolecular Cloning lPolymerase Chain Reaction (PCR) lRestriction Enzymes Example: EcoR：5-G A A T T C -3 3-C T T A A G -5 Sma I : 5- CCC GGG -3 3- GGG CCC-5lVectors Example: plasmid(5000bp), bacteriophage lambda(20kb)，BAC(100-300kb), YAC(1000-2000kb)Molecular CloningThe process

5、 of cloning a segment of human DNA into a plasmid cloning vectorBacteriophage VectorsThe Polymerase Chain ReactionPCR: Enzymatic amplification of a fragment of DNA located between a pair of primersThe Polymerase Chain ReactionThe use of molecular cloning and the PCR to isolate large quantities of a

6、particular desired DNA sequence of interest in pure formConstruction of LibrarieslGenomic LibrarieslComplementary DNA libraries Construction of a “library” of DNA from the human genome in a bacteriophage vectorConstruction of a cDNA library in a plasmid vectorLibrary ScreeninglNucleic Acid Probes Pr

7、obes: a cloned DNA or RNA molecule, labeled with radioactivity or another detectable tracer, used to identify its complementary sequences by molecular hybridizationlNucleic Acid HybridizationThe principle of nucleic acid hybridizationMETHODS OF NUCLEIC ACID ANALYSISlSouthern BlottinglAnalysis with A

8、llele-Specific Oligonucleotide (ASO) ProbeslNorthern BlottingThe southern blotting procedureDetection of single base-pair mutations using ASO probes Fluorescence In Situ Hybridization (FISH)lPrinciplelProbes: three typeslgene-specific or locus-specific probes, lrepetitive DNA probes (satellite DNA p

9、robes) lprobes for entire chromosomes or chromosome armstwo-color, three-color, multicolorlApplicationThe FISH in which fluorescently tagged DNA probes were hybridized to human metaphase chromosome Alpha satellite centromere probe-FISH Detection of telomeres at the ends of each chromosome by fluores

10、cence in situ hybridization using a repeated TTAGGG probe. In Situ Hybridization to ChromosomeslFluorescence In Situ Hybridization (FISH) Spectral Karyotyping (SKY) Comparative Genome HybridizationSpectral Karyotyping (SKY)Analysis of metaphase chromosomes from a prostate cancer cell line SKYG-bands

11、In Situ Hybridization to ChromosomeslFluorescence In Situ Hybridization (FISH) Spectral Karyotyping (SKY) Comparative Genome Hybridization Fluorescently labelled normal DNA Fluorescently labelled tumour DNARatio of red-to-green represent situation of chromosome Sanger SequencingDNA SEQUENCE ANALYSIS

12、METHODS OF PROTEIN ANALYSISlWestern BlottingWestern blot demonstrating absence of the protein dystrophin in protein extracts from DMD pateintsHow to apply the tools in medical genetics Detection of genetic polymorphisms Linkage AnalysislGenetic polymorphism: The alleles are found in more than 1% of

13、chromosomes in the general population.l Rare variants: 1% of chromosomes of the general populationApplications of Polymorphisms in Medical GeneticslMapping a gene to a particular region of a chromosome by linkage analysislPrenatal diagnoses of genetic diseaselDetection of heterozygous carrier of gen

14、etic diseaselRFLP (Restriction Fragment Length Polymorphisms) DNA-based variations at restriction sites detected by Southern blotting RFLPs detected by DNA hybridization (Southern Blotting)Codominant inheritance of an X-linked RFLPMinisatellite and Microsatellite PolymorphismslVNTR (variable number

15、of tandem repeat） Insertion of multiple copies of a DNA sequence 10 to 100 base pairs in the DNA between two restriction site, known as a minisatellite.Codominant inheritance of a autosomal DNA polymorphism caused by VNTR Minisatellite and Microsatellite PolymorphismslMicrosatellite Markers Microsat

16、ellite are stretches of DNA consisting of repeating units of two, three or four nucleotides, such as T G T G T G , C A A C A A C A A , o r AAATAAATAAAT. GENE MAPPINGlPhysical Mapping of Human GeneslGenetic Mapping of Human Genes by Linkage Analysis Physical mapping Assigning a gene to a particular b

17、and of particular chromosome and its relationship to other genes in terms of numbers of base pairsMethods:n Somatic cell hybridsn FISHn Genomic Sequencing Somatic Cell Hybrids Mixtures of cell types from two same or different specieMonochromosomal assignmentRegional assignment Somatic Cell Hybrids E

18、xtinction Isolate cells from affected patient Make cell lines Make hybrids Retain hybrids with the disease phenotype Identify choromosomesSomatic cell hybridsRandom loss of human chromosomesMapping Human Genes by Linkage AnalysislLinkage The tendency for alleles close together on the same chromosome

19、 to be transmitted together,as a intact unit, through meiosislGenetic Linkage Analysis A method of mapping genes that uses family studies to determine whether two genes show linkage when passed on from one generation to the nextLinkageRecombinationMeiotic behavior of alleles at two loci are on the s

20、ame chromosomeThe smaller the recombination frequency, the closer together two loci are Genetic MarkerslRFLPs, microsatellites, STR (short tandem repeats)，et.allMarker - segment of recognizable DNAlMarker does not necessarily code for something functionallEach chromosome has several marker identifie

21、dlCan start assigning functional genes to locations relative to the markers STRACACACPrimer 1Primer 2ACACACACACACACACACACACACACACAC132412345IIIIII1 211221, 22, 53, 42, 32, 3Lod Scores (Z) L( 0.5)nZ = log10 L( = 0.5)n Z 3 is considered definitive evidence that two loci are linked( equivalent to great

22、er than 1000:1 odds in favor of linkage) n Z 1 suggest that the two loci are linkedn Z 1 suggest that the two loci are unlinkedTechnologies used in gene therapyvAntisense RNA expression vectorvDirect injection of antisense RNA/DNAvAntisense RNA delivered by liposomevOthersTdRdTMPdTDPdTTP1TKHSV1TKHSV

23、cytosol TKcytosol dTMP-Kcytosol dNDP-KGCVGCVMPGCVDPGCVTP1TKHSV1TKHSVcytosol dNDP-KA gene that transforms a nontoxic form of a drug (pro-drug) into a toxic substance. vherpes simplex virus thymidine kinase gene (HSV1TK) Suicide GeneThe application of RNAi to the medical geneticsRNA interference (RNAi

24、)In 1990 . In the petunia “cosuppression”In 1994. In the fungi “quelling”In 1995. In C.elegans sense RNA can block the expression of par-1 geneIn 1998. Fire and Mello suggested new mechanism for the phenomenon of gene silencing-RNAi The phenomenon of RNA inference - What is RNAi ? RNA interference (

25、RNAi) RNAi leading to the selective degradation of target mRNA and knockdown of gene function is induced by introducing into cells a short dsRNA called short interfering RNA or siRNA.Remarkable Properties of RNAi dsRNA (not ssRNA) is interfering agent Sequence-specific loss of mRNA and protein Effec

26、tive against exons not introns Potent (few dsRNA molecules are effective) Persistent (affects next generation) Effects can cross cell barriers (feed, soak)Mechanism of RNAiTwo step mechanistic model:1.Initiation stepGeneration of siRNAs2.Effective stepDegradation of target mRNA Amplified mechanistic

27、 : 1. Primate siRNA- dsRNAs were cut by Diser 2. Sub siRNA- produced dsRNAs by RdRP were cut Diser into new siRNA Initiation StepATPATPADP + ppiADP + ppiEffective Stepv Genome-wide RNAi screens Invertebrates have proved useful in high-throughput RNAi screens. Apply global RNAi screens to ageing-rela

28、ted disorders are likely to be of considerable benefit. RNAi screens is high specificity,but must be confirmed using complementary techniques.v Gene silencing Gene silencing is nature response to invasive virus in the plant-RNA-mediated anti-virus immunoreaction. transcriptional gene silencing,TGS p

29、ost- transcriptional gene silencing, PTGS RNAi? 1. Long dsRNA induces non-specific response of mammalian 2. 21nt siRNA-induced gene silencing has high specificityvsiRNA Therapeutics RNAi-mediated gene knockdown coupled with the advantages of using model organisms offers many opportunities for the de

30、velopment of new therapies. infection by virus : hepatitis , HIV various cancers: CDK2 genetic disease : SBMA and polyglutamine disorder. poly(Q) diseasev Regulate the expression of gene RNAi plays important role in the epigenetics. siRNA effects on the form of the chromic. It can direct difference of cells.lMuscular DystrophylSpinal Muscular