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Gene Mapping

Gene Mapping

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  • 1. GENE MAPPINGGENE MAPPING By A.Arputha SelvarajBy A.Arputha Selvaraj
  • 2. Comparative GenomicsComparative Genomics  Gene functions have been conservedGene functions have been conserved across evolutionacross evolution  Nature solves a problem, it rarely solves itNature solves a problem, it rarely solves it again.again.
  • 3. Eyeless Mutation http://www.ucm.es/info/genetica/AVG/practicas/Drosophila/Drosophila.htm
  • 4. Drosophila Eyeless Human Aniridia Homologous Genes 50% identical
  • 5. Human gene expressed in correct location in fly carrying the mutant eyeless gene http://www.geo.de/GEO/fotografie/portfolio_des_monats/2001_10_portfolio_meckes/page2.html?SDSID=
  • 6. Ectopic Expression of Human Eyeless Gene in Drosophila http://as3.lib.byu.edu/~imaging/brad/set5/sl17.html
  • 7. Flies with human eyeless homolog do not develop “human” eyes
  • 8. Develop compound eyes, even with human homolog driving development
  • 9. Normal Eyeless Gene Developmental end product depends on context Human Fruit fly
  • 10. Normal Eyeless Gene A B D E
  • 11. Evolution & GenesEvolution & Genes  The function of many genes is conserved acrossThe function of many genes is conserved across immense evolutionary distances.immense evolutionary distances.  The function of a gene is affected by its environment.The function of a gene is affected by its environment.  Many gene products function as part of a cascade orMany gene products function as part of a cascade or pathway.pathway.
  • 12. “Nothing in Biology Makes Sense Except in the Light of Evolution” (Theodosius Dobzhansky)
  • 13. Map Genes in the Context ofMap Genes in the Context of ChromosomesChromosomes
  • 14. Uses of Gene MappingUses of Gene Mapping  Identify genes responsible for diseases.Identify genes responsible for diseases.  Heritable diseasesHeritable diseases  CancerCancer  Identify genes responsible for traits.Identify genes responsible for traits.  Plants or AnimalsPlants or Animals  Disease resistanceDisease resistance  Meat or Milk ProductionMeat or Milk Production
  • 15. Types of MapsTypes of Maps  Nucleotide Sequence MapsNucleotide Sequence Maps  complete or partially sequenced organismscomplete or partially sequenced organisms  Cytogenetic MapsCytogenetic Maps  Breakpoints in diseaseBreakpoints in disease  Direct binding of probes to chromosomeDirect binding of probes to chromosome  Genetic Linkage MapsGenetic Linkage Maps  MarkersMarkers  Physical MapsPhysical Maps
  • 16. Mapping GenesMapping Genes  Essential element -- MarkersEssential element -- Markers  Differences between two members of aDifferences between two members of a species.species.  Typically between 1-400 nucleotides inTypically between 1-400 nucleotides in length.length.  Can also be gross chromosomalCan also be gross chromosomal rearrangements.rearrangements.
  • 17. DNA SequencingDNA Sequencing  Two methods originally developed:Two methods originally developed:  Chemical or Maxim and Gilbert method.Chemical or Maxim and Gilbert method.  Dideoxynucleotide or Sanger method.Dideoxynucleotide or Sanger method.  Both Gilber and Sanger won Nobel prizes forBoth Gilber and Sanger won Nobel prizes for developing these techniques.developing these techniques.  Dideoxynucleotide method is by far theDideoxynucleotide method is by far the most common employed today.most common employed today.
  • 18. Automated SequencingAutomated Sequencing  Each nucleotide is labeled with aEach nucleotide is labeled with a different fluorescent chromophore.different fluorescent chromophore.  Uses Taq polymerase to incorporateUses Taq polymerase to incorporate fluorescent dideoxy nucleotide.fluorescent dideoxy nucleotide.  Analysis on a polyacrylamide gel orAnalysis on a polyacrylamide gel or capillary matrix that is can separatecapillary matrix that is can separate DNA based upon a 1 nucleotideDNA based upon a 1 nucleotide difference.difference.  30-1000 nucleotides in length can30-1000 nucleotides in length can theoretically be determined.theoretically be determined.
  • 19. Deoxy Dideoxy OH O O Base CH2OP O O O Base CH2OP O O O (3’2’)
  • 20. Copyright Cold Spring Harbor Laboratories
  • 21. G A T C G G A T C C G A T C G G A T C C T A G G
  • 22. G A T C G G A T C C T A G GCC dd G A T C G G A T C C T A G GCC dd
  • 23. G A T C G G A T C C T A G GCCG dd
  • 24. Nucleotide SequencingNucleotide Sequencing  Complete Genome SequencingComplete Genome Sequencing  Bacterial Artificial Chromosomes (BAC)Bacterial Artificial Chromosomes (BAC) • Can grow in bacteriaCan grow in bacteria • Have large inserts 100,000-300,000Have large inserts 100,000-300,000 nucleotidesnucleotides  Collect a large library of BACsCollect a large library of BACs  Restriction Endonuclease (RE) MapRestriction Endonuclease (RE) Map • RE - cuts DNA at specific sitesRE - cuts DNA at specific sites • EcoRI cuts at GAATTCEcoRI cuts at GAATTC
  • 25. meds.queensu.ca/~mbio318/ EXTRA_MATERIAL.html B A C s
  • 26. Genomic SequencingGenomic Sequencing  Once BAC map completeOnce BAC map complete  Sequence ends of BAC clones.Sequence ends of BAC clones.  With dense BAC map, large regions may beWith dense BAC map, large regions may be covered by overlapping the sequences.covered by overlapping the sequences.  Individual BACs can be completelyIndividual BACs can be completely sequenced.sequenced.  Individual BACs can be hybridized toIndividual BACs can be hybridized to chromosomes to identify chromosome ofchromosomes to identify chromosome of origin.origin.
  • 27. RecombinationRecombination  Permits Mapping by providing:Permits Mapping by providing:  Linkage groupsLinkage groups  DistancesDistances  During MeiosisDuring Meiosis
  • 28. RecombinationRecombination  Linkage Groups - markers that tend toLinkage Groups - markers that tend to remain together.remain together.  Distance - the further apart two markersDistance - the further apart two markers lie, the more often recombination willlie, the more often recombination will occur between those markers.occur between those markers.  Markers on the same chromosome can beMarkers on the same chromosome can be so far apart that they appear in differentso far apart that they appear in different linkage groups.linkage groups.
  • 29. Cytogenetic MappingCytogenetic Mapping Philadelphia ChromosomePhiladelphia Chromosome  Chronic myelogenous leukemia (CML) isChronic myelogenous leukemia (CML) is characterized by a reciprocal translocationcharacterized by a reciprocal translocation between chromosomes 9 and 22 thatbetween chromosomes 9 and 22 that produces the Philadelphia chromosome.produces the Philadelphia chromosome. Invariably there is disease progression,Invariably there is disease progression, with loss of the capacity for terminalwith loss of the capacity for terminal differentiation by the hematopoietic stemdifferentiation by the hematopoietic stem cell, resulting in an acute leukemia.cell, resulting in an acute leukemia.  www.clubstewart.co.uk/cml.htmwww.clubstewart.co.uk/cml.htm
  • 30. Genetic Linkage MapsGenetic Linkage Maps PolymorphismPolymorphism  PolymorphismsPolymorphisms  Polymorphism - an allele present in aPolymorphism - an allele present in a population that exhibits multiple forms.population that exhibits multiple forms.  Monomorph - Single form in a population.Monomorph - Single form in a population.  Common.Common.  Typically result in no effect on survival ofTypically result in no effect on survival of individual.individual.
  • 31. Types of MarkersTypes of Markers  Single Nucleotide Polymorphisms (SNPs)Single Nucleotide Polymorphisms (SNPs)  Occur approximately every 100-300 bp inOccur approximately every 100-300 bp in humans.humans. Names: rs8111765
  • 32. MarkersMarkers  Microsatellites or Tandem RepeatsMicrosatellites or Tandem Repeats  acgCACACAtgcacgCACACAtgc  acgCACACACAtgcacgCACACACAtgc
  • 33. Physical MapsPhysical Maps  Sequence Tagged Sites (STSs)Sequence Tagged Sites (STSs)  Use Polymerase Chain Reaction (PCR).Use Polymerase Chain Reaction (PCR).  Primers based upon:Primers based upon: • random sequencerandom sequence • expressed sequence tagexpressed sequence tag  Amplify from library of clones containingAmplify from library of clones containing large inserts (BAC).large inserts (BAC).  Relate to BAC map.Relate to BAC map.  If more than one on same clone, then closeIf more than one on same clone, then close togethertogether
  • 34. Types of questionsTypes of questions  Where does gene X exist within the genome of organism Y?Where does gene X exist within the genome of organism Y? What are some flanking markers?What are some flanking markers?  Which genes exist on a chromosome, and in what order do theyWhich genes exist on a chromosome, and in what order do they appear?appear?  Show the genes that exist in region R of the chromosome. ShowShow the genes that exist in region R of the chromosome. Show me the corresponding sequence data for that region.me the corresponding sequence data for that region.  Display the region of a chromosome between points A and B.Display the region of a chromosome between points A and B.  Show both the cytogenetic and sequence map for that region,Show both the cytogenetic and sequence map for that region, aligned to each other based on markers that have been placedaligned to each other based on markers that have been placed on both maps.on both maps.  What is the distance between two genes? (Note: scale dependsWhat is the distance between two genes? (Note: scale depends on the type of map on which those genes have been placed.)on the type of map on which those genes have been placed.)  I know the cytogenetic location of gene X. What is theI know the cytogenetic location of gene X. What is the corresponding physical location?corresponding physical location? http://www.ncbi.nlm.nih.gov/mapview/static/MapViewerHelp.html
  • 35. Genes and MapsGenes and Maps  Bcl2Bcl2  Map LinkMap Link  OMIM LinkOMIM Link  Syntenic Relationships - tendency ofSyntenic Relationships - tendency of closely linked genes to remain linkedclosely linked genes to remain linked during evolution.during evolution.
  • 36. GO evidence codesGO evidence codes
  • 37. PathwaysPathways  KEGG pathwaysKEGG pathways Examine Cell Adhesion MoleculesExamine Cell Adhesion Molecules -What does JAM3 appear to interact with?-What does JAM3 appear to interact with? -What information is available if you click on-What information is available if you click on JAM2?JAM2? What motifs are found in JAM2?What motifs are found in JAM2? Click on Tight JunctionClick on Tight Junction -What molecules appear to interact with the JAM--What molecules appear to interact with the JAM- JAM dimer? What is ZO-1JAM dimer? What is ZO-1
  • 38. Thank YouThank You