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Gene Mapping
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GENE MAPPINGGENE MAPPINGBy A.Arputha SelvarajBy A.Arputha Selvaraj
Comparative GenomicsComparative Genomics
Gene functions have been conserved Gene functions have been conserved across evolutionacross evolution
Nature solves a problem, it rarely solves it Nature solves a problem, it rarely solves it again.again.
Eyeless Mutation
http://www.ucm.es/info/genetica/AVG/practicas/Drosophila/Drosophila.htm
Drosophila Eyeless
Human Aniridia
Homologous Genes50% identical
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=
Ectopic Expression of Human Eyeless Gene in Drosophilahttp://as3.lib.byu.edu/~imaging/brad/set5/sl17.html
Flies with human eyeless homologdo not develop “human” eyes
Develop compound eyes, even with human homolog driving development
Normal Eyeless Gene
Developmental end product depends on
context
Human
Fruit fly
Normal Eyeless Gene
A
B
D
E
Evolution & GenesEvolution & Genes
The function of many genes is conserved across The 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 or Many gene products function as part of a cascade or pathway. pathway.
“Nothing in Biology Makes Sense Except in the Light of Evolution” (Theodosius Dobzhansky)
Map Genes in the Context of Map Genes in the Context of ChromosomesChromosomes
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
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
Mapping GenesMapping Genes
Essential element -- MarkersEssential element -- Markers Differences between two members of a Differences between two members of a
species.species. Typically between 1-400 nucleotides in Typically between 1-400 nucleotides in
length.length. Can also be gross chromosomal Can also be gross chromosomal
rearrangements.rearrangements.
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 for Both Gilber and Sanger won Nobel prizes for
developing these techniques.developing these techniques. Dideoxynucleotide method is by far the Dideoxynucleotide method is by far the
most common employed today.most common employed today.
Automated SequencingAutomated Sequencing Each nucleotide is labeled with a Each nucleotide is labeled with a
different fluorescent chromophore.different fluorescent chromophore. Uses Taq polymerase to incorporate Uses Taq polymerase to incorporate
fluorescent dideoxy nucleotide.fluorescent dideoxy nucleotide. Analysis on a polyacrylamide gel or Analysis on a polyacrylamide gel or
capillary matrix that is can separate capillary matrix that is can separate DNA based upon a 1 nucleotide DNA based upon a 1 nucleotide difference.difference.
30-1000 nucleotides in length can 30-1000 nucleotides in length can theoretically be determined.theoretically be determined.
Deoxy
Dideoxy
OH
O
O
Base
CH2OP
O
O
O
Base
CH2OP
O
O
O
(3’2’)
Copyright Cold Spring Harbor Laboratories
G A T C G G A T C C
G A T C G G A T C C
T A G G
G A T C G G A T C C
T A G GCCdd
G A T C G G A T C C
T A G GCCdd
G A T C G G A T C C
T A G GCCGdd
Nucleotide SequencingNucleotide Sequencing
Complete Genome Sequencing Complete Genome Sequencing Bacterial Artificial Chromosomes (BAC)Bacterial Artificial Chromosomes (BAC)
• Can grow in bacteriaCan grow in bacteria• Have large inserts 100,000-300,000 Have 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 sites RE - cuts DNA at specific sites • EcoRI cuts at GAATTC EcoRI cuts at GAATTC
meds.queensu.ca/~mbio318/ EXTRA_MATERIAL.html
BACs
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 be With dense BAC map, large regions may be covered by overlapping the sequences.covered by overlapping the sequences.
Individual BACs can be completely Individual BACs can be completely sequenced.sequenced.
Individual BACs can be hybridized to Individual BACs can be hybridized to chromosomes to identify chromosome of chromosomes to identify chromosome of origin.origin.
RecombinationRecombination Permits Mapping by providing:Permits Mapping by providing:
Linkage groupsLinkage groups DistancesDistances
During MeiosisDuring Meiosis
RecombinationRecombination
Linkage Groups - markers that tend to Linkage Groups - markers that tend to remain together.remain together.
Distance - the further apart two markers Distance - the further apart two markers lie, the more often recombination will occur lie, the more often recombination will occur between those markers.between those markers.
Markers on the same chromosome can be Markers on the same chromosome can be so far apart that they appear in different so far apart that they appear in different linkage groups.linkage groups.
Cytogenetic MappingCytogenetic MappingPhiladelphia ChromosomePhiladelphia Chromosome
Chronic myelogenous leukemia (CML) is Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation characterized by a reciprocal translocation between chromosomes 9 and 22 that between 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 terminal with loss of the capacity for terminal differentiation by the hematopoietic stem differentiation 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
Genetic Linkage Maps Genetic Linkage Maps PolymorphismPolymorphism
PolymorphismsPolymorphisms Polymorphism - an allele present in a Polymorphism - 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 of Typically result in no effect on survival of
individual.individual.
Types of MarkersTypes of Markers Single Nucleotide Polymorphisms (SNPs)Single Nucleotide Polymorphisms (SNPs)
Occur approximately every 100-300 bp in Occur approximately every 100-300 bp in humans.humans.
Names: rs8111765
MarkersMarkers
Microsatellites or Tandem RepeatsMicrosatellites or Tandem Repeats acgCACACAtgcacgCACACAtgc acgCACACACAtgcacgCACACACAtgc
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 containing Amplify 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 close If more than one on same clone, then close
togethertogether
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 they Which genes exist on a chromosome, and in what order do they appear? appear?
Show the genes that exist in region R of the chromosome. Show Show 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 placed aligned 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 depends What 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 the I 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
Genes and MapsGenes and Maps
Bcl2Bcl2 Map LinkMap Link OMIM LinkOMIM Link Syntenic Relationships - tendency of Syntenic Relationships - tendency of
closely linked genes to remain linked closely linked genes to remain linked during evolution.during evolution.
GO evidence codesGO evidence codes
PathwaysPathways
KEGG pathwaysKEGG pathwaysExamine 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
Thank YouThank You