说明2013版理工课件cloning gwas

1、Positional cloningMain goal of genetics:link phenotypes with genotypesCopyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 92GenotypePhenotypeChromosomeGeneAllelePolymorphismGenetic markerInheritanceLinkageDNA sequenceEpistasisDomina

2、nceGene expression Tracing InheritanceDetection of genetic variants:-causative mutations direct variants-genetic markers“Evaluation” of Inheritance-linkage studies-association studies Manifestation of phenotypeMendelian traitNon-mendelian traits-Sex linked plex traitsExtensive allelic variation dist

3、inguishes individuals within a speciesDefinition of a locus any location in the genome that is defined by chromosomal coordinatesCan have multiple genes or no genesCan be a single base pair or millions of base pairsDefinition of an allele any variation in the DNA sequence, even if it doesnt have an

4、effect on phenotypeDefinition of polymorphism -Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 114Genetic markerA genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify in

5、dividuals or species-must be identifiable through inheritance polymorphic- representing an alleleRestriction site-altering SNPs detected by Southern blotsIn this example, the SNP affects an EcoRI restriction siteAllele 1 has an EcoRI site that is not present in allele 2After digestion with EcoRI, th

6、e two SNP alleles produce different-sized DNA fragmentsCopyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 115Fig. 11.4Genetic variation can be caused by subtraction or addition of short sequencesDeletion-insertion polymorphisms

7、 Short insertions or deletions of a single or a few base pairsAlso called InDelsSimple sequence repeats (SSRs)One-, two-, or three-base sequences repeated 15-100 times in tandemArise because of stutter of DNA polymerase during replication of the repeat sequences Copyright The McGraw-Hill Companies,

8、Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 116Microsatellite1-5 nucleotides repeats-generated by DNA slippage-arise one every 1000 gamete(SNP 1/109 )-frequent enough to provide good info-not frequent enough to arise in several generations even in large fam

9、iliesDeletions or duplications of a DNA regionMinisatellites 20-100 bp long repeats repreated up to 1000 times _ unit sizes are 500 bp to 20 kbRepeat sequences occur at multiple genomic loci Detected using restriction digests and hybridization of Southern blots with cross-hybridizing minisatellite p

10、robeIdeal for DNA fingerprintingCopyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 118Minisatellite analysis provides a broad comparison of whole genomesCopyright The McGraw-Hill Companies, Inc. Permission required to reproduce

11、 or display Hartwell et al., 4th edition, Chapter 119Fig. 11.12(a) Digest DNA with restriction enzyme that does not cut inside minisatellite(b) After electrophoresis, perform Southern blotting and hybridize with probe containing minisatellite sequenceHow many minisatellite loci have to be examined i

12、n order to prove identity?DNA fingerprinting developed by Alec Jeffreys in 1985Probability of two individuals with identical genotypes at loci with two equally prevalent alleles One locus, probability = 37.5%10 unlinked loci, probability = 37.510 = 0.005% (1 in 20,000)24 unlinked loci, probability =

13、 37.524 = 1 in 17 billionTotal human population = 8 billionTherefore, if 24 minisatellite loci analyzed, there is virtually no chance of two different individuals (except identical twins) having identical genotypesCopyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display

14、Hartwell et al., 4th edition, Chapter 1110Distribution of SNPs, InDel, and SSR variantsin the 400 kb that contains the CFTR regionAverage frequency over the entire human genome:SNPs, one every 1kbInDels (DIPs), one every 10 kbCA-repeat SSR, one every 30 kb Copyright The McGraw-Hill Companies, Inc. P

15、ermission required to reproduce or display Hartwell et al., 4th edition, Chapter 1111Fig. 11.8bGenetic MarkerA landmark that identifies a unique location on a chromosomeA gene or other identifiable portion of DNA whose inheritance can be followed A polymorphic genetic property that can be used to di

16、stinguish the parental origin of allelesBecause DNA segments that lie near each other on a chromosome tend to be inherited together, markers are often used as indirect ways of tracking the inheritance pattern of a gene that has not yet been identified, but whose approximate location is known Tracing

17、 InheritanceDetection of genetic variants:-direct variants-genetic markersEvaluation of Inheritance-linkage mapping-association studies Manifestation of phenotypeMendelian traitNon-mendelian traits-Sex linked plex traitsDatabases of human Mendelian traitsYearNumber of entries198652198779198814219892

18、851990417199170919921161199313421994231619952339199629651997361819984485199951462000527320015552200249402003441820041329Total4656816144625690115125Total2267021502115Other, mainly phenotypes withsuspected mendelian basis1478041321342%Mendelianphenotypeorlocus,molecular basis unknown17652521411597# Ph

19、enotype description,molecular basis known4030035368+ Gene with known sequenceand phenotype1023137484439703* Gene with known sequenceTotalMitochondrialY-LinkedX-LinkedAutosomalClassification of 923 genetic disordersby age of onset Majority (39%) of disorders is caused by genetic alterations of enzyme

20、sHigh proportion of enzymes likely reflects a high proportion of a total number of enzymes in the genome of higher eukaryotes (25-30%) ?Next (14%) is proteins modulating function of other proteins: activation, stabilization, folding of a second proteinDiseases caused by enzymes of all functional cat

21、egories appear in all ages of onsetEven higher overrepresentation of enzymes is observed in first year of age. During the fetus development the mothers homeostatic environment might compensate for discrepancies caused by an enzymatic disorder which will manifest immediately after birth Example:Aldol

22、ase B, fructose-bisphosphatase =Fructose intoleranceClassification of 923 genetic disordersby age of onset Mutations in transcription factors are overrepresented in in utero onset which likely indicates their essential role in development Example:Homeo box-A13Hand-foot-uterus (HFU) syndrome is chara

23、cterized by abnormalities of the hand, foot, urinary tract, and reproductive tract.Classification of 923 genetic disordersby age of onset Classification of 923 genetic disordersby age of onset Modulators of protein function related diseases have a late onset as their effect is subtle and slowly accu

24、mulates throughout lifeExample:Parkin - ubiquitin ligase protein degradationParkinsons disease - accumulation of neurodegenerative proteins “waste products” in neuronsEnzymesProtein modifiersReceptorsTranscriptionfactorsClassification by disease characteristicsMost diseases in all categories are ver

25、y rare or rare1. Vast majority of enzyme caused disease are autosomal recessive which reflects that mutations typically deactivate an enzyme and both alleles need to be deactivated. Typical onset in 1st year of life; in utero mother metabolism compensates and masks the traitIn receptors, recessive a

26、nd dominant are split and in transcription factors is mostly dominant; mutation introduces an undesired activity; TF AD is even more dominant due to gain of an undesired function; TFs are essential for development affecting embryonic development the most123EnzymesProtein modifiersReceptorsTranscript

27、ionfactorsClassification by disease characteristicsDifferent peaks in onset:-TF in utero-Enzymes within 1 year (both see previous slide)1. Receptors in puberty reflects an importance of receptors (signaling between cells and tissues) in rapid growth and development2.-Protein modifiers later in life

28、reflects a slow progression of the disease, protein modifiers do not affect the metabolism dramatically and their effect is slow and cumulative12Complex genetic traitsA measurable phenotype, such as disease status or a quantitative character, that is influenced by many environmental and genetic fact

29、ors Genetic modifiers of cystic fibrosisMutations in CFTR almost always cause CF phenotype; broad variation in phenotype manifestations which depends on mutation in modifier genes Obstructions in small and large intestinesCFM CF modifier geneThe most common phenotype is modified by at least four gen

30、esA simple Mendelian trait is essentially nonexistent amongst genetic traits in human as well as model organisms (mouse). Mutations in nitric oxide synthase modify microbial infections in lungsComplexities that alter traditional Mendelian ratiosMost human traits dont have single-gene inheritance pat

31、ternsLinkage mapping and positional cloning can be done with complex traits, but it is more difficult and the mapping strategy is different from single-gene traitsCopyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 1125Table 11.

32、2Complex genetic traitToday, 1 in 166 individuals is diagnosed with autism, making it more common than pediatric cancer, diabetes, and AIDS combined. Autism is a neurobehavioral spectrum of phenotypes characterized by deficits in the development of languageand social relationships and patterns of re

33、petitive, rigid and compulsive behaviors.Whole genome screen linkage findings for autismspectrum disorders. The peak linkage findings from nine independentand four follow-up studies are illustrated. MLS maximum multipoint lod score (evaluating multiple makers together).Differential linkage of variou

34、s loci to endophenotypes of Autism Spectrum Disorder (ASD)Obsessive compulsive phenotype1q24.46q14.319p13Endophenotype hereditary characteristic that is normally associated with some condition but is not a direct symptom of that condition Differential linkage of various loci to endophenotypes of Aut

35、ism Spectrum Disorder (ASD)Obsessive compulsive phenotype1q24.46q14.319p13Endophenotype hereditary characteristic that is normally associated with some condition but is not a direct symptom of that condition “Developmental milestones”17q11.219.p13Differential linkage of various loci to endophenotype

36、s of Autism Spectrum Disorder (ASD)Obsessive compulsive phenotype1q24.46q14.319p13Endophenotype hereditary characteristic that is normally associated with some condition but is not a direct symptom of that condition “Developmental milestones”17q11.219.p13Developmental regression7q36.121q21.1Challeng

37、es for nowadays geneticsMajor challenges for future genetic studies-traits with extremely low frequencies-difficult phenotypic evaluations plex genetic traitsContribution of the human (and other) genome project(s)-more genetic markers (SNP)-genome-wide association studies (GWAS)Functional genomics:

38、dynamic properties of genes -gene expression and gene function-model organisms (yeast drosophila, worm, mouse)-epigenetics and gene regulationSingle nucleotide polymorphisms (SNPs)SNPs account for the vast majority of total sequence variation between humans genomes 18 million human SNPs have been identifiedArise from rare mistakes in replicationPer-base mutation rate is 1 in 30 million per generationEach SNP can be traced back to a genome change that occurred in a single ancestral genomeNotations for SNP genotypesCopyright The McGraw-Hil