基因诊断与基因治疗概论课件gene_d_t_08-3(1).ppt

基因诊断与基因治疗概论（3）,Gene Diagnosis and Gene Therapy,An Introduction to,Fan LemingAthrosclerosis Research CenterNanjing Medical University,CONTENTS,内 容,Introduction Why do gene diagnosis and therapyConception What is gene diagnosis and therapyMethodology How do gene diagnosis and therapyClinical Applications,Introduction Why do gene diagnosis and therapyConception What is gene diagnosis and therapyMethodology How do gene diagnosis and therapyClinical Applications,Part I Gene Diagnosis, How do gene diagnosis and therapy,Methodology,方法学：基因诊断,Disease is related not only to the structure of gene but also to the transcription or translation levelGene diagnosis may put in practice by: 1. DNA diagnosis 2. RNA diagnosis 3. Fluorescence in situ Hybridization (FISH) 4. Gene chip,to analyze the structure of gene,to measure the expression of gene,to detect deletion of large fragment,to detect multiple mutation simultaneously,基因诊断常用方法,Disease is related not only to the structure of gene but also to the transcription or translation levelGene diagnosis may put in practice by: 1. DNA diagnosis 2. RNA diagnosis 3. Fluorescence in situ Hybridization (FISH) 4. Gene chip,基因诊断常用方法,Gene diagnosis,1,The abnormal structure of gene may be detected at DNA level directly. If the pathogenic gene or its structure and the details of mutation is uncertain, it is necessary to judge the existence of pathogenic gene indirectly by means of genetic markers. Such means is called Linkage analyses. The infectious disease may be diagnosed by detecting the exogenous DNA of pathogen.,1. DNA Diagnosis,DNA诊断,Direct detection of known gene mutation Restriction fragment analysis, etc.,The abnormal structure of gene may be detected at DNA level directly. If the pathogenic gene or its structure and the details of mutation is uncertain, it is necessary to judge the existence of pathogenic gene indirectly by means of genetic markers. Such means is called Linkage analyses. The infectious disease may be diagnosed by detecting the exogenous DNA of pathogen.,Linkage Analysis Restriction fragment length polymorphism,1. DNA Diagnosis,DNA诊断,The abnormal structure of gene may be detected at DNA level directly. If the pathogenic gene or its structure and the details of mutation is uncertain, it is necessary to judge the existence of pathogenic gene indirectly by means of genetic markers. Such means is called Linkage analyses. The infectious disease may be diagnosed by detecting the exogenous DNA of pathogen.,1. DNA Diagnosis,DNA诊断,Detection of exogenous DNA,1. DNA Diagnosis,DNA诊断,Direct detection of gene mutation 1、Restriction fragment analysis 2、Oligonucleotide dot blot analysis 3、Amplification refractory mutation system 4、Single-strand conformation polymorphism 5、 DNA sequencing,Detection of exogenous DNA,Linkage Analysis Restriction fragment length polymorphism,1. DNA Diagnosis,DNA诊断,Direct detection of known gene mutation 1、Restriction fragment analysis 2、Oligonucleotide dot blot analysis 3、Amplification refractory mutation system 4、Single-strand conformation polymorphism 5、 DNA sequencing,Detection of exogenous DNA,Linkage Analysis Restriction fragment length polymorphism,To judge the existence of known mutation. Be applicable to cases in which the pathogenic gene is certain and its sequence is complete or partial known.,（1）Direct detection of gene mutation,Common techniques： 1) Restriction fragment analysis Be applicable to larger fragment mutation or point mutation located in restrictive enzyme site 2) Oligonucleotide dot blot hybridization analysis Be applicable to various point mutation, particularly outside of the restrictive enzyme site.,突变的直接检测,1) Restrict fragment analysis,Larger fragment deletion,Point mutation located in restrictive enzyme site,限制性片断分析,1.15kb,1.35kb,bA/ bS,bA,bS,MstII CCTNAGG,Sickle Cell Anaemia: Point mutation in coden 6 of b-globin GAG(Glu) GTG(Val),Example 1. Point mutation which leads to loss or gain of restrictive enzyme site,限制性片断分析：例 1,Homo,Hetero,Normal,5.2kb,4.6kb,Example 2. Larger fragment mutation which leads to restrictive enzyme site shift,b-Thalassemia ： 0.6kb deletion at 3end of b-globin,| | BgI II BgI II 4.6kb,| | BgI II BgI II 5.2kb,限制性片断分析：例 2,a a 14kb,Example 3. Gene deletion,a-Thalassemia ： various number (1-4) of b-globin gene deletion,a,10kb,14kb,10kb,aa/aa aa/- aa/a- a-/- -/-,BamHI,BamHI,限制性片断分析：例 3,Combination of alleles,1/1,1/2,1/3,1/4,2/2,2/3,2/4,3/3,3/4,4/4,Example 4. Repeat sequence,Southern blot,Allele 1,Allele 4,Allele 3,Allele 2,限制性片断分析：例 4,CCGGC CCGTC HpaII,x,138bp,If the sequence around restrictive enzyme site is known, the simple and convenient method is PCR-RFLP analysis.,e.g. Point mutation at coden 12 of H-ras gene： GGC-GTC,38bp 100bp,PCR分析限制性位点,Genomic DNA or PCR product hybridized with ASO probe,Be applicable to detect known point mutation,2) Oligonucleotide dot hybridization blot analysis,Incomplete hybridyzation,ASO: Allele Specific Oligonucleotide,寡核苷酸斑点杂交,Example: Sickle Cell Anaemia Point mutation in coden 6 of b-globin GAG(Glu) GTG(Val) bA probe ACTCCTGA GGAGAAGTCTGCC bS probe ACTCCTGT GGAGAAGTCTGCC,Hetero,bA probe,bS probe,Homo,Normal,寡核苷酸斑点杂交：实例,Reverse Dot Hybridization，RDH Be applicable in disease with multiple mutational site,probe 1 2 3 4,反向斑点杂交,3) amplification refractory mutation system,NN NN,control,AC AC1 AC AC1 AC AC1 BD BD1 BD BD1 BD BD1,300bp,250bp,MM MM,MN MN,control,300bp,250bp,扩增阻滞突变体系,Protocol ： Normal Fragment Mutant Fragment,4) Single-strand conformation polymorphism,PCR-SSCP analysis is applicable to explore uncertain point mutation in pathogenic gene, which may downsize the detective scope into an exon or an fragment and final sequencing to confirm mutation site.,单链构象多态性分析,Amplify region of interest,Denature samples in Formamide and Heat,DNA molecules conformation depends on their sequence,Run on a Non-denaturing gel and look for mobility differences,单链构象多态性分析,The detective result for exon 13 in LDL receptor gene of Family D,D4 D3 D1 D2 N N,单链构象多态性分析；实例,5) PCR-sequencing method,PCR测序分析：实例,1. DNA Diagnosis,DNA诊断,Direct detection of gene mutation 1、Restriction fragment analysis 2、Oligonucleotide dot blot analysis 3、Amplification refractory mutation system 4、Single-strand conformation polymorphism 5、 DNA sequencing,Detection of exogenous DNA,Linkage Analysis Restriction fragment length polymorphism,Linkage analysis: Take certain pathogenic gene linked polymorphism as genetic marker , to explore existence of pathogenic gene in members of same family.,（2） Linkage analyses,遗传连锁分析,Genetic linkage: Two or over gene or site located closely within one chromosome and to be co-segregation (inherited together),DNA polymorphism mark system: Restriction Fragment Length Polymorphysm (RFLP) Most of RFLPs do not represent a mutation but a linkage mark EcoR1 Sites Southern blot,three different molecular genotypes,polymorphic site,*,(About 100,000 RFLPs in human genome),DNA多态标记：RFLP, Microsatellite markers Variable Number of Tandem Repeats ( VNTRs ) 24bp Short Tandem Repeats (STR),three different molecular genotypes,polymorphic sequences PCR products,(At least 650,000 STRs in human genome),DNA多态标记：微卫星,ATGGTAAGCCTGAGCTGACTTAGCGTATGGTAAACCTGAGTTGACTTAGCGT snp snp, Single nucleotide polymorphysms (SNPs),Single base-pair differences between individuals are pretty common and easy to find in contrast to RFLPs and VNTRs (There are 1,400,000 SNPs in human genome, about 1 in 1000bp),DNA多态标记：SNP,1）Key member (parents) should be heterozygous in order to differentiate homologous chromosome； 2）There should be homozygous in filial generation in order to confirm pathogenic gene and its genetic marker 3）Parental generation to be examined must be biological.,?,连锁分析要点,Linkage analysis is performed within family：,proband,Proband 3.7/3.7(+/+),连锁分析存在问题：正确性,(-/+),(-/+),Fetus Probability Diagnosis Validity 8.0/3.7 (-/+) 50 Normal or Homozygous 8.0/8.0 (-/-) 25% Heterozygous 3.7/3.7 (+/+) 25% Heterozygous,Proband 8.0/3.7(/+),50,连锁分析存在问题：正确性,(-/+),(-/+),Proband 3.7/3.7(+/+),连锁分析存在问题：正确性,(+/+),(-/+),Allele linked-RFLP (The allele linked with single polymorphism site) e.g. The first antenatal diagnosis for haemophilia by chorion villus sampling (CVS),连锁分析：实例 1,PCR-RFLP linkage anylysis,连锁分析：实例 2,1. PCR product, undigested 2. PCR from CVS, Bcl I digested 3. PCR from father, Bcl I digested 4. PCR from mother, Bcl I digested,1. DNA Diagnosis,DNA诊断,Direct detection of gene mutation 1、Restriction fragment analysis 2、Oligonucleotide dot blot analysis 3、Amplification refractory mutation system 4、Single-strand conformation polymorphism 5、 DNA sequencing,Detection of exogenous DNA,Linkage Analysis Restriction fragment length polymorphism,Be applicable in infectious disease e.g. virus, bacteria, fungi, rickettsia, mycoplasma, chlamydia May detect the pathogen directly from clinical samples by molecular hybridization (specific gene probe) or PCR (specific primer) Will be more early and quick, more sensitive and distinctive as compare to conventional microbiological, immunological or serological diagnosis.,（3） Detection of exogenous DNA,检测外源DNA：病原体,Disease is related not only to the structure of gene but also to the transcription or translation levelGene diagnosis may put in practice by: 1. DNA diagnosis 2. RNA diagnosis 3. Fluorescence in situ Hybridization (FISH) 4. Gene chip,基因诊断常用方法,Gene diagnosis,2,To detect the transcriptional ability, efficiency and its products (mRNA, usually by RT-PCR) （1） mRNA quantitative analysis （2） Differential display PCR（DD-PCR） （3）RNA length analysis,2. RNA Diagnosis,RNA诊断常用方法,DD-PCR (mRNA differential display) Total mRNA 12 of 3 anchor primer （T11MA、T11MG、T11MC、T11MT） Reverse transcriptase 12 of cDNA 1st strand 2426 of 5 random primer run 288312 of PCR with 1st strand Taq polymerase cDNA double strands (May obtain all cDNA of mammal cell),差异显示逆转录PCR,Normal tissue,Diseased tissue, Total mRNA Total mRNA ss cDNA ds cDNA ds cDNA ss cDNA amplified product amplified product PAGE PAGE autoradiography autoradiography Cut out special band Cut out special band Elution Elution PCR amplification PCR amplification sequencing data-base sequencing screening new EST YAC screening chromosome locating,差异显示逆转录PCR,Diseased tissue,Normal tissue,Disease is related not only to the structure of gene but also to the transcription or translation levelGene diagnosis may put in practice by: 1. DNA diagnosis 2. RNA diagnosis 3. Fluorescence in situ Hybridization (FISH) 4. Gene chip,基因诊断常用方法,Gene diagnosis,3,Probe：labeled with nonradioactive biotin or digoxinDetective system：fluorescein coupled Ag-Ab,Has been extensively applied in congenital malformation, tumor and prenatal diagnosis.,Differentiate number alteration and structural aberration of chromosome,Determine the