genetics maps. genotyping individuals with str’s
TRANSCRIPT
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Genetics Maps
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Genetics Maps
• Genotyping individuals with STR’s
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Genetics Maps
• By 1994, there was a ~1 cM map based largely on microsatellites (STR’s)
A comprehensive human linkage map with centimorgan density. Murray JC, Buetow KH, Weber JL, Ludwigsen S, Scherpbier-Heddema T, Manion F, Quillen J, Sheffield VC, Sunden S, Duyk GM, et al.
Science. 1994 Sep 30;265(5181):2049-54.
5840 loci total3617 polymerase chain reaction-formatted short tandem repeat polymorphisms427 genes0.7 centimorgan density
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Genetics Maps
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Genetics Maps
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Physical Maps
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Physical Maps
Ordering clones based on Hybridization
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Physical Maps
Ordering clones based STS content
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Genetic Maps and Physical Maps are Aligned by STS
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Sequencing the Human Genome
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Gel run
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Sequencing the Human Genome
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Sequencing the Human Genome
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Sequencing the Human Genome
• Sequence ~ 500 bp each reaction– To sequence the Human genome,
sequencing method needs to be: • FAST• CHEAP
– In 1990 reality was:
• SLOW• EXPENSIVE (>$1 per base!)
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Sequencing the Human Genome
• International Human Genome Sequencing Consortium– Primarily six institutes with high-throughput
sequencing capabilities• Whitehead Institute• The Sanger Center• Washington University• DOE Sequencing Center• Bayer College of Medicine (31 Jan 2005 161,489 kb; 2,935,479 kb)
• In 1990, the IHGSC began a 15 year plan to sequence the entire Human genome
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Sequencing the Human Genome
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Sequencing the Human Genome
• IHGSC Strategy - Shotgun sequencing of ordered BAC contigs
• Define BAC contig order based on STS
• Sequence each cluster of BAC’s within contig – align based on sequence
• Anchor to genome by STS….
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Sequencing the Human Genome
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Sequencing the Human Genome
• In 1998, Celera Genomics announced plans to sequence the human genome…
• …175,000 sequence reads per day, operating 24 hours a day, 7 days a week
J. Craig Venter
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Sequencing the Human Genome
• Whole genome shotgun approach vs. Clone by Clone approach
• By-passes the initial work of ordering clones
• Celera performed about 32 million sequence reads, each 500 – 1000 bp
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Sequencing the Human Genome
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Sequencing the Human Genome
• IHGSC published sequence reads every 24 hours to prevent patenting of DNA
• Celera had access to IHGSC data
• Debate over whether Celera could have shotgun sequenced the genome without IHGSC data
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Sequencing the Human Genome
• Both groups published results simultaneously
• Celera – Science
February 2001
• IHGSC – Nature
February 2001
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Sequencing the Human Genome
Nature 409, 818 - 820 (15 February 2001)
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Sequencing the Human Genome
• Controversy! Science published Celera’s sequence without requiring deposition to GenBank
• Celera provides full access, with a catch…
• Celera provided Science with a copy in escrow
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Sequencing Your Human Genome
• For $500,000 you can have your DNA sequenced
• Sequence 1000 individual human genomes
• “Personalized” medicine
J. Craig Venter
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• Next Gen Sequencing
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• The proliferation of genetic testing resources
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Human Genome
• Legal considerations– Should DNA, or genes, be patentable?
• In the past, USPTO considered genes as man-made chemicals– Copy DNA region, splice it together, and
propagate it in bacteria, etc
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Human Genome
• Celera >6500 genes
• Human Genome Sciences >7000
• Incyte >50,000
• Only a fraction may be awarded by USPTO, and only a fraction of these may be useful in treating human disease
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Human Genome
• 1994 U. of Rochester scientists isolate mRNA for COX-2 and clone gene
• Suggest that compounds which inhibit COX-2 might provide pain relief from arthritis
• Submit patent application in 1995
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Human Genome
• 1998 – Celebrex – inhibitor of cyclooxygenase-2 (COX-2) introduced as arthritis medication
• Developed by Pfizer/Searle
• Development began in early-90’s i.e. around time of U. of Rochester discovery
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Human Genome
• April 2000, U. of Rochester awarded patent covering COX-2 gene and inhibition of the peptide product thereof
• The same day, U. of Rochester files lawsuit against Pfizer/Searle to block Celebrex sales
• Claims that Pfizer/Searle infringes on their patent
• They want royalties from the sale of the invention
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Human Genome
• 2003 – U. of Rochester patent found invalid
• 2004 – Invalidation upheld by higher Court
• U. of Rochester patent did not provide sufficient example of what the inhibitor would be…i.e. claims too broad without a working example
• How will “basic science” performed by Universities be rewarded?
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Human Genome
• Vioxx and Celebrex in news again this year: increased risk of “cardiovascular event” i.e. heart attacks
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Human Genome
• Gene discovery– Average gene extends over 27 kb – Average 8.8 introns– Average 145 bp
• Extremes:– Dystrophin gene 2.4 Mb– Titin gene contains 178 introns, coding for a
80,780 bp mRNA
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Human Genome
• Gene discovery– One approach is to examine “transcriptome”– Exome
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Human Genome
• Conservation of chromosome/gene location between organisms
• Synteny
• Exons tend to be conserved between species
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Human Genome
• Human vs. Pufferfish genome
• Pufferfish genome about 1/7th the size of the human genome with similar number of genes
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Human Genome
• Predictive computer programs, e.g. GENSCAN
• GENSCAN predicts the location of genes based on splicing predictions, promoter regions and other criteria
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Human Genome
• Online databases have formed to curate Human genome data
• Ensembl (www.ensemble.org)
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Genetic Mapping of Mendelian Characters
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Identifying Disease-Causing Gene Variations
• Linkage analysis and Positional Cloning
– Clone disease gene without knowing anything except the approximate chromosomal location
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Recombination
• Recombination during meiosis separates loci– More often when they are farther apart– Less often when they are close
• Recall discussion of the Genetic Map– Loci on separate chromosomes segregate
independently– Loci on the same chromosome segregate as a
function of recombination
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Recombination
13-1
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13_06.jpg
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Linkage analysis
• Linkage analysis locates the disease gene locus
– Linkage analysis requires• Clear segregation patterns in families• Informative markers close to the locus
– Utilize LOD analysis to verify linkage– Calculate cM distance between Loci
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Positional Cloning
• Widely used strategy in human genetics for cloning disease genes
• No knowledge of the function of the gene product is necessary
• Strong for finding single-gene disorders
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Positional Cloning
• Linkage analysis with polymorphic markers establishes location of disease gene
• LOD score analysis, and other methods are employed
• Once we know the approximate location…– The heavy molecular biology begins
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Positional Cloning
• Example - Huntington’s disease
– CAG…
– Autosomal dominant– 100% penetrance– Fatal– Late onset means patients often have children
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Finding the Huntington Gene – 1981-1983
• Family with Huntington's disease found in Venezuela
• Originated from a single founder - female
• Provided:– Traceable family pedigree– Informative meiosis
– Problem was… only a few polymorphic markers where known at the time
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Finding the Huntington Gene
• Blood samples taken
• Check for disease symptoms
• Paternity verified
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Finding the Huntington Gene
• By luck, one haplotype segregated very closely with Huntington disease
• Marker was an RFLP called G8 (later called D4S10)
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Finding the Huntington Gene
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Finding the Huntington Gene
• Locate the region to the tip of the short arm of chromosome 4 by linkage with G8 (D4S10)
• Maximum LOD score occurred at about 4 cM distance, i.e. 4 in 100 meiosis
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Finding the Huntington Gene
• Together this started an international effort to generate YAC clones of the 4 Mb region
• More polymorphisms were found
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Finding the Huntington Gene
• Next, find an unknown gene in an uncharacterized chromosome location
• Locate CpG islands
• Cross-species comparisons
• Further haplotype analysis suggested a 500 Kb region 3’ to D4S10
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Finding the Huntington Gene
• Exon trapping was key
• Compare cloned exons between normal and Huntington disease patients
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Finding the Huntington Gene
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Finding the Huntington Gene
• One exon, called IT15, contained an expanded CAG repeat….
• Mapping to 4 cM – 1983
• Cloning of Huntington gene – 1993
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Complex Disease and Susceptibility
Gene
Gene
Disease
Single gene disorders
Mendelian Inheritance
High penetrance
Low environmental influence (but sometimes significant)
LOD-based linkage analysis works great
Genetic heterogeneity
Low population incidence
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Complex Disease and Susceptibility
Gene
Gene
Gene
Gene
Environment
Disease A Disease B Disease C
Multifactorial disorders