Download Mapping the Human Genome - Scheid Signalling Lab @ York

Genetics Maps
Genetics Maps
• Genotyping individuals with STR’s
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 total
3617 polymerase chain reaction-formatted short tandem repeat polymorphisms
427 genes
0.7 centimorgan density
Genetics Maps
Genetics Maps
Physical Maps
Physical Maps
Ordering clones based on Hybridization
Physical Maps
Ordering clones based STS content
Genetic Maps and Physical Maps
are Aligned by STS
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3’
Sequencing the Human Genome
5’
Gel run
Sequencing the Human Genome
Sequencing the Human Genome
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!)
Sequencing the Human Genome
• International Human Genome Sequencing
Consortium
– Primarily six institutes with high-throughput
sequencing capabilities
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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
Sequencing the Human Genome
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….
Sequencing the Human Genome
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
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
Sequencing the Human
Genome
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
Sequencing the Human
Genome
• Both groups published results simultaneously
• Celera – Science
February 2001
• IHGSC – Nature
February 2001
Sequencing the Human
Genome
Nature 409, 818 - 820 (15 February 2001)
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
Sequencing Your Human Genome
• For $500,000 you can have your DNA
sequenced
• Sequence 1000 individual human
genomes
• “Personalized” medicine
J. Craig Venter
• Next Gen Sequencing
• The proliferation of genetic testing
resources
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
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
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
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
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
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?
Human Genome
• Vioxx and Celebrex in news again this
year: increased risk of “cardiovascular
event” i.e. heart attacks
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
Human Genome
• Gene discovery
– One approach is to examine “transcriptome”
– Exome
Human Genome
• Conservation of chromosome/gene
location between organisms
• Synteny
• Exons tend to be conserved between
species
Human Genome
• Human vs. Pufferfish genome
• Pufferfish genome about 1/7th the size of the
human genome with similar number of genes
Human Genome
• Predictive computer programs, e.g.
GENSCAN
• GENSCAN predicts the location of genes
based on splicing predictions, promoter
regions and other criteria
Human Genome
• Online databases have formed to curate
Human genome data
• Ensembl (www.ensemble.org)
Genetic Mapping of Mendelian
Characters
Identifying Disease-Causing
Gene Variations
• Linkage analysis and Positional Cloning
– Clone disease gene without knowing anything
except the approximate chromosomal location
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
Recombination
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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
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
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
Positional Cloning
• Example - Huntington’s disease
– CAG…
– Autosomal dominant
– 100% penetrance
– Fatal
– Late onset means patients often have children
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
Finding the Huntington Gene
• Blood samples taken
• Check for disease symptoms
• Paternity verified
Finding the Huntington Gene
• By luck, one haplotype segregated very
closely with Huntington disease
• Marker was an RFLP called G8 (later
called D4S10)
Finding the Huntington Gene
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
Finding the Huntington Gene
• Together this started an international effort
to generate YAC clones of the 4 Mb region
• More polymorphisms were found
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
Finding the Huntington Gene
• Exon trapping was key
• Compare cloned exons between normal
and Huntington disease patients
Finding the Huntington Gene
Finding the Huntington Gene
• One exon, called IT15, contained an
expanded CAG repeat….
• Mapping to 4 cM – 1983
• Cloning of Huntington gene – 1993
Complex Disease and
Susceptibility
Single gene disorders
Gene
Mendelian Inheritance
High penetrance
Low environmental influence (but
sometimes significant)
Gene
LOD-based linkage analysis works great
Genetic heterogeneity
Disease
Low population incidence
Complex Disease and
Susceptibility
Gene
Gene
Gene
Gene
Environment
Disease A
Disease B
Disease C
Multifactorial disorders