ch7(1) gene hunting and isolation-基因組學(xué)

1、GenomicsChapter 7 Gene hunting and function research(1) Gene hunting and transcript identificationGenomicsFunction research of large genome1Gene hunting:How to Find valuable genes?2Get expressed sequences of genesHow?3Research on the functions of genesHow?GenomicsHow to find valuable genes?From geno

2、me sequencesFrom chromosome locationFrom specific proteinFrom mRNA/cDNA differencesGenomicsMajor Strategies for gene huntingFunctional cloningPhenotype cloningPositional cloningPositional candidate cloningUsing information about the function of a known protein that could be involved in a genetic dis

3、ease. This approach has very limited application. Large scale mutation by transposon tagging (轉(zhuǎn)座子標(biāo)簽法)Gene expression differences analysisUsing only information about the genes approximate chromosomal location obtained from gene mapping.Using information from map position and the genes possible funct

4、ion, homology, and expression pattern. This approach has been quite successful and will dominate other strategies.Genomics7.1 Gene hunting7.1.1 Functional cloning7.1.2 Transposon tagging7.1.3 Positional cloning*7.1.4 Positional candidate cloningGenomics7.1.1 Functional cloningvMethods: Purifying spe

5、cific proteinsequencing the N-/C-end amino aciddesign DNA probeshybridizing with DNA/cDNA clone librarygain the destination gene Purifying specific proteinantibody screening in cNDA expression library Phage display (p212) vDisadvantages Sufficient purified protein Specificity of antibodyGenomicsExam

6、ple: gene clone for Factor VIIIGenomics7.1.2 transposon tagging of plant geneParents cross（one with active transposon）Selection mutant for specific traitConstruction genomic library of mutantsScreen the library with transposon probeSubclone the flanking sequences of transposonScreen the genomic libr

7、ary of normal plant by the probes of subcloned sequencesGain clones of destination geneflo gene isolation from Antirrhinum by transposon taggingGenomicsLimitations of transposon taggingvUseful for the plants owning endogenous transposon such as rice, maize, Arabidopsis thaliana(擬南芥), potato, Antirrh

8、inum(金魚草) etc.vLow mutation frequency leads to heavy workload.vMutation phenotype should be visible. Genomics7.1.3 Positional cloning (map-based cloning)v After a halting start in the mid1980s, the number of disease genes succumbing to cloning efforts based solely on pinpointing their position in th

9、e genome is growing exponentially. More than 40 genes have been identified so far. Collis F S. Positional cloning moves from perditional to traditional, Nature Genet, 1995,9 (4): 347-350.v Genes associated with inherited disease were identified by positional cloning including Alzheimers disease, bre

10、ast cancer, and polycystic kidney diseaseGenomicsComparison of positional and functional cloningGenomicsWhat is positional cloning?vThe core problem for positional cloninggene localization.vExpression of genes position on chromosome: Cytogenetic locationdescribe the rough position on chromosome. Mol

11、ecular locationA genes molecular address pinpoints the location of that gene in terms of base pairs.vMethods to localization gene Cytogenetic analysis Genome scan using molecular markers GenomicsCytogenetic locationv Numberingv P（短臂）v q （長臂）v Ter/telv CenGenomicsGenetic diseasesv Single gene (or Men

12、delian) disorder Autosomal dominant disorderBrachydactyly(短指/趾畸形) Autosomal recessive disorderalbinism (白化病) X-linked dominant disordervitamin D resistant rickets (抗VD佝僂病) X-linked recessive disorderDuchenne muscular dystrophy Y-linked disorderHairy ears Mitochondrial disorderv Polygenic diseases A

13、genetic disease caused by the combined actions of two or more genes. higher incidence rate(發(fā)病率) Such as coronary heart disease(冠心病), diabetes(糖尿病), hypertension, cleft lip(唇裂) and peptic ulcers(胃潰瘍). They are not inherited in simple Mendelian patterns.GenomicsExample for early state gene localizatio

14、n: Duchenne muscular dystrophy (DMD) genev Duchenne muscular dystrophy (DMD) is the most common of the childhood muscular dystrophies, affecting 1 in 3,500 males worldwide. Duchenne MD gets worse over time, and boys usually lose the ability to walk by age 12, and generally dont survive beyond their

15、20s due to cardiomyopathy.v Inheritance: typical sex-linked recessive disorder affecting only boys.v Dystrophin: The largest human gene (2500Kb, 79 exons, 14Kb cDNA, 427KD protein)GenomicsHow to hunt DMD gene?Backgrounds vIt is a single gene disease.vIt is a X-linked gene. vNo pedigree.vA specific m

16、ale individual(B.B.) with 3 X-linked diseasesDMD, retinitis pigmentosa(RP,色素性視網(wǎng)膜炎) and chronic granulomatous disease(CGD,慢性肉芽腫) has an apparent interstitial(縫隙) deletion in the short arm of X.GenomicsHow to hunt DMD gene?Methods v Phenol-accelerated competitive DNA reassociation Sheared DNA from B.B

17、. by sonication to 1 kb. Mbo Icleaved DNA from a 49, XXXXY human lymphoid cell line.v Molecular cloning of appropriately reassociated molecules.v 4/81 clones were found absent from the male patients DNA. v The 4 clones were localized, on the basis of hybridization with restriction nuclease-digested

18、genomic DNA from a panel of human and human-rodent hybrid cell lines, into three regions surrounding band 21 of the short arm of the normal human X chromosome.200-fold excessiveThree types of molecularterminiGenomicsHow to hunt DMD gene?resultsv Autoradiograph of the hybridization of two different P

19、ERT library clones to various HindIII-cleaved genomic DNA samples.v The clone labeled p4B12 hybridizes to a 2.5 kb HindIII fragment in all samples.v pERT87 hybridizes to a 1.1-kb HindIII fragment in 1,2,3,5 DNA samples.rodent-human hybrid cell DNA sample retaining human X chromosome from Xp11.3Xqter

20、.GenomicsLocalization of DMD gene患者染色體的缺失部分雜合細(xì)胞中人染色體的保留部分Reference: Specific Cloning of DNA Fragments Absent from the DNA of a Male Patient with an X Chromosome. Kunkel LM et.al. PNAS,1985;82;4778-4782 GenomicsDMD基因治療的爭論基因治療的爭論v 2004年7月，我國中山大學(xué)附屬第一醫(yī)院完成全球首例干細(xì)胞移植治療假肥大型肌營養(yǎng)不良癥（DMD基因缺陷）？v FIRST U.S. TRIAL

21、 OF DMD GENE THERAPY UNDER WAY：COLUMBUS, Ohio, March 29, 2006 The first U.S. human gene therapy trial directed at was launched yesterday at Columbus (Ohio) Childrens Hospital, the Muscular Dystrophy Association (MDA), Childrens Hospital, and Asklepios Biopharmaceutical Inc. (AskBio) announced today.

22、 GenomicsHow to localize the gene on chromosomevCytogenetic abnormalityvGenome scanning (全基因組掃描) : Looking for the markers closest to the disease gene. (采用DNA分子多態(tài)性標(biāo)記，以較大間距在大量樣本、家系或同胞對中進(jìn)行全基因組掃描，通過連鎖分析將相關(guān)基因定位到某些染色體區(qū)域；在這些區(qū)域再選擇高密度的遺傳標(biāo)記，做精細(xì)分析，進(jìn)一步縮小定位區(qū)域；查找該定位區(qū)域內(nèi)的所有基因，從中選擇可能的候選基因進(jìn)行基因變異檢測。)v 3200Mb/24=133Mb

23、GenomicsPrinciple steps of positional cloningGenomics3 factors in gene location by genome scanvSamplevGenetic DNA polymorphism markers RFLP markers Microsatellite or STR markers Single nucleotide polymorphism (SNP)vStatistic methods Linkage analysis (連鎖分析) Association analysis (關(guān)聯(lián)分析)GenomicsGenome s

24、canning analysis methods Linkage analysis Big pedigree Sufficient DNA markers Mathematic methods Log odds of score method, LOD(對數(shù)優(yōu)勢記分法) Allele sharing method (等位基因法) Association analysis Diseased individuals without consanguinity Younger populations Whole genome scan with STRs or SNPs More useful in

25、 gene hunting for polygenic diseasesGenomicsGenome scanning analysis methods2 major methodsAssociation analysisDiseased and control individualsNo inheritance parametersDiscover linkage disequilibrium (LD, 連鎖連鎖不平衡）不平衡）Marker: STR and SNPMore precise More useful in gene hunting for polygenic diseasesL

26、inkage analysisBig pedigreeLOD method Need inheritance parametersDiscover linkage between markers and diseaseMarker: STR precision1cMMathematic methods Log odds of score method, LOD(對數(shù)優(yōu)勢記分法) Allele sharing method (等位基因法)GenomicsWhat is linkagevLinkage: Linkage describes the phenomenon whereby allele

27、 at neighboring loci are close to one another on the same chromosome, they will be transmitted together more frequently than chance. Recombination fraction number of recombinant gametes/total gametes Recombination fraction is a measure of genetic distance. 1cM = 1%chance of recombination between two

28、 loci.GenomicsLinkage studiesv Linkage studies look for co-segregation of marker alleles and disease within pedigrees: Parametric linkage analysis (Lod score method): extended pedigrees(家系) with specified disease model. Non-parametric linkage analysis: allele sharing between pairs of affected relati

29、ves, no disease model specified.v For complex diseases, there may be many genes influencing risk as well as the effects of the environment.v Much more difficult to collect pedigrees with multiple affecteds, and no guarantee of the same (or any) disease gene segregating in the family. GenomicsLod Sco

30、revLod得分是在一定重組率下兩個位點相連鎖的似然性與兩個位點不連鎖的似然性比值的對數(shù)值 L( 3: evidence of linkage 2 lod score 3: suggestive evidence of linkage -2 lod score 2: uninformative of linkage lod score -2: exclusion of linkageGenomicsLimitations of Lod Score Method vassumes single locus inheritance vrequires specification of diseas

31、e gene frequency and penetrance(外顯率) vhas reduced power when disease model is grossly misspecifiedGenomicsLod Score Method SoftwarevLINKAGE vFASTLINK vVITESSE vGENEHUNTERv-Example: Gene of Brachydactyly(短指/趾畸形) Type A-1(student report)GenomicsMapping a disease locus(Autosomal dom)phenotype (variatio

32、n in locus 1)marker genotype (variation in locus 2)A1A2GenomicsMapping a disease locus(Autosomal dom)phenotype (variation in locus 1)A1A2GenomicsMapping a disease locus(Autosomal dom)phenotype (variation in locus 1)marker genotype (variation in locus 2)A1A2GenomicsMapping a disease locus(Autosomal d

33、om)phenotype (variation in locus 1)marker genotype (variation in locus 2)How close are they in genetic distance?A1A2GenomicsMapping a disease locusA1DA2d(Autosomal dom)A1A2GenomicsMapping a disease locusA1DA2d(Autosomal dom)A1A2(assume phase)GenomicsMapping a disease locusA1DA2dA1dA1dA1A2GenomicsMap

34、ping a disease locusA1DA2dA1dA1dA1A2A2dA1dGenomicsMapping a disease locusA1DA2dA1dA1dA1A2A1DA1dGenomicsMapping a disease locusA1DA2dA1dA1dA1A2A2dA1dGenomicsMapping a disease locusA1DA2dA1dA1d?A1A2GenomicsMapping a disease locusA1dA1d?A1DA2d(sperm)A1A2GenomicsMapping a disease locusA1dA1dA2DA1DA2d(sp

35、erm)A1A2A1dGenomicsMapping a disease locusWhat is the apparent RF between the DNA marker and the disease mutation?A. 1/10B. 1/8C. 1/20A1A2GenomicsMapping a disease locusWhat is the apparent RF between the DNA marker and the disease mutation?A. 1/10B. 1/8C. 1/201/8 = 12.5 m.u.A1A2GenomicsMapping a di

36、sease locusWe now know the mutation is near (linked to) the marker.1/8 = 12.5 m.u.A1A2GenomicsMapping a disease locusWe now know the mutation is near (linked to) the marker.marker (known)1/8 = 12.5 m.u.A1A2GenomicsMapping a disease locusWe now know the mutation is near (linked to) the marker.window

37、containing causative mutation1/8 = 12.5 m.u.marker (known)A1A2GenomicsMapping a disease locusWhat is the distance between the two boundaries of the window?A.1/8 m.u.B.12.5 m.u.C. 50 m.u.D. 25 m.u.markerwindow containing causative mutation1/8 = 12.5 m.u.A1A2GenomicsMapping a disease locus1/8 = 12.5 m

38、.u.How significant?A1A2GenomicsMapping a disease locus1/8 = 12.5 m.u.How significant?If RF = 0.5 (unlinked), would be like flipping a coin 8 times.How likely would you be to get 7 heads and 1 tail?A1A2GenomicsIf RF = 0.5 (unlinked), would be like flipping a coin 8 times.How likely would you be to ge

39、t 7 heads and 1 tail?How much MORE likely is a model of RF 0.5?GenomicsIf RF = 0.5 (unlinked), would be like flipping a coin 8 times.How likely would you be to get 7 heads and 1 tail?How much MORE likely is a model of RF 6 times more likely under LINKED hypothesis than under UNLINKED hypothesis.k =

40、1 recomb, n = 7 non-recomb.GenomicsLOD scoresrodds0.112.2440.210.7370.36.3250.42.8670.5?Odds = P(pedigree | r)P(pedigree | r = 0.5)Odds = (1-r)n rk 0.5(total # meioses)k = 1 recomb, n = 7 non-recomb.GenomicsLOD scoresOdds = P(pedigree | r)P(pedigree | r = 0.5)Odds = (1-r)n rk 0.5(total # meioses)Odd

41、s at r=0.5?A.2.5B.0C. 1D. 10rodds0.112.2440.210.7370.36.3250.42.8670.5?GenomicsLOD scoresWhats the best (most likely) value of r?A.0.1B.0.2C. 0.3D. 0.4E.0.5rodds0.112.2440.210.7370.36.3250.42.8670.51GenomicsLOD scoresOnly believe r 0.5 (truly linked) when odds 1000!rodds0.112.2440.210.7370.36.3250.4

42、2.8670.51GenomicsLod mapping0.00.10.20.30.40.5Lod-3.92.71.50.50.0GenomicsCystic fibrosis mapping, 1985(via somatic cell hybrid mapping)Nature 318, 384 - 385 (28 November 1985)GenomicsCystic fibrosis mapping, 1985GenomicsHaplotype association studies for high-resolution mapping in humansvHaplotypes a

43、re sets of closely linked alleles. Specific combination of two or more DNA marker alleles situated close together on the same DNA molecule Usually SNPs Distance between SNPs on a haplotype must be short enough that they stay associated during transmission over many generationsGenomicsFormation of ha

44、plotypes over revolutionary timeGenomicsAncient disease loci are associated with haplotypesGenomicsAssociation analysisv Disease-marker association studies look for correlation between disease and marker alleles by making use of the larger pedigree that is the population(種群): more recombination in s

45、hared ancestry (useful for fine-scale mapping); unknown relationships between all individuals;v Proposed as powerful alternative to linkage studies for complex diseases: will depend on availability of high-density map of markers; unlikely to be useful for detecting extremely rare variants.GenomicsLi

46、nkage Disequilibrium (LD)vWhat is Linkage Disequilibrium (連鎖不平衡） Linkage disequilibrium is a term used in the study of population genetics for the non-random association of alleles at two or more loci, not necessarily on the same chromosome. P(disease & M) P(disease) x P(M)GenomicsLD of linkageG

47、enomicsHaplotype tagging SNPs and Haploview GenomicsReasons for LDvReasons for LD Linkage Genetic drift (遺傳漂變) Natural selection (自然選擇) Founder effect (始祖效應(yīng)) GenomicsCharacters of sample affecting associationvInitiation sizevGenerationsvPopulation stratification(層化)vPopulation stabilityGenomicsGene

48、hunting of polygenic diseasesv Polygenic inheritance: Two or more genes interact in the expression of phenotype. (Cardiovascular disease, Hypertension, etc.) quantitative trait loci (QTLs) Unlimited number of transmission patterns for QTLs Discrete traits penetrance may increase with number of mutan

49、t loci Expressivity may vary with number of loci Many other factors complicate analysis Some mutant genes may have large effect Mutations at some loci may be recessive while others are dominant or codominant GenomicsAssociation study for polygenic diseasev Start with population genetically isolated

50、for a long time such as Icelanders or Amishv Collect DNA samples from subgroup with diseasev Also collect from equal number of people without diseasev Genotype each individual in subgroups for haplotypes throughout entire genomev Look for association between haplotype and disease phenotypev Associat

51、ion represents linkage disequilibriumv If successful, provides high resolution to narrow parts of chromosomesGenomicsExample Alzheimers and APOEAllele E4 in APOE(載脂蛋白) is approximately three times more frequent in Alzheimers patients than healthy controls.Genomics7.1.4 Positional candidate cloningGe

52、nomicsDiagram for positional (candidate) cloningGenomicsSummary of positional candidate cloning(1)1Find extended families in which disease is segregating2Use panel of polymorphic markers spaced at 10 cM intervals (300 STRs) across all chromosomes3Determine genotype for all individuals in families fo

53、r each DNA markerGenomicsSummary of positional candidate cloning(2)4Look for linkage between a marker and disease phenotype5Once region of chromosome has been narrowed down to 1Mb or less, all genes within are identified.6Candidate genes: Usually about 17 genes per 1Mb fragment; Identify coding regi

54、onsGenomics7.2 Transcript identification (p188195)7.2.1 ORF scanning*7.2.2 Hybridization test7.2.3 cDNA sequencing7.2.4 Exon trappingGenomics7.2.1 ORF scancomputer methodsv The coding regions of genes are ORFs (Open Reading Frames，開放閱讀框). Initiation codon: ATG, GTG, TTG Termination codon: TAA, TAG,

55、TGAv Each double-stranded DNA molecule has 6 reading frames.v Length parameter (usually 100 codons)GenomicsFeasibility of ORF scanningv Simple ORF scanning is an effective way of locating genes in a bacterial genome.v Simple ORF scanning is less effective with higher eukaryotic DNA.Why? Introns Non-

56、coding intergenic sequencesGenomicsModifications in ORF scanning software vCodon bias (密碼子偏愛) Codon bias refers to the fact that not all codons are used equally frequently in the genes of a particular organism. vExon-intron boundaries(外顯子-內(nèi)含子邊界) Upstream (donor site): 5-AGGTAAGT-3 Downstream (accept

57、or site): 5-PyPyPyPyPyPyNCAG-3vUpstream regulatory sequences(上游調(diào)控序列): promoter etc.vCpG island in vertebrate genome內(nèi)含子的5端稱供體位，3端稱受體位。GenomicsCodon bias in human genomeAmino acidcodonusage AlaGCAGCCGCGGCT22%41%11%26% ThrACAACCACGACT27%38%12%23% ValGTAGTCGTGGTT11%0.5%48%17%*reference from GenomicsHomo

58、logy searches assist ORF inspectionv Two types of homologous sequences Paralogs(旁系同源) and orthologs(直系同源)v Manifestation of homology: Similarity in DNA sequences Similarity in AA sequences: domain, motif Similarity in protein tertiary structurev Homology searches assist ORF inspection If the tentati

59、ve exon sequence gives one or more positive matches after a homology search then it is probably a real exon; If it gives no match then its authenticity must remain in doubt until it is assessed by one or other of the experiment-based gene location techniques.Genomics7.2.2 Hybridization testvHybridiz

60、ation tests can determine if a fragment contains transcribed sequences. Northern blotting Zoo blottingGenomicsNorthern blottingradioactive RNA hybridizes only to its complementary sequencedry and expose to X-ray film Gel electro-phoresis of total mRNA (agarose)GenomicsWhat Northern blotting can tell us?