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What geneticists do:
From Baxevanis/Ouellette, p. 142:
Most researchers utilize genome
information in one of three ways:
• To find out what genomic elements are
contained within a genomic region
• To determine the order of defined
elements within a region
• To determine the chromosomal position of
a particular element
A Timeline of The Human
Genome
# human genes mapped to
a chromosome location
# years it would take to
sequence the human genome
• 1967
none
sequencing not possible yet
• 1977
3 genes mapped
4,000,000 years to
finish at 1977 rate
• 1987
12 genes mapped
1000 years to finish
at 1987 rate
• 1997
30,000 genes mapped
50 years to finish
Finding Genes that cause
Disease?
• Does completion of the HGP signal the end
of the ‘gene hunt’?
• Will the completion of the human genome
sequencing lead to the immediate
identification of all disease genes?
The STS map:
• STS = sequence-tagged site.
• STS are short, unique fragments of DNA generated
by PCR.
• Verification of a human STS: PCR amplification of
the human genome generates one small fragment 
unique landmark.
Usefulness of STSs
STSs are used in various mapping
activities:
• STSs are used to find overlaps between
fragments of genomic DNA.
• Finding overlaps  ordering of fragments
(see handout).
Utility of STSs:
• STSs would be more valuable if
they were assigned to
chromosomes and ordered.
• STSs are often ordered by
Radiation Hybrid Mapping.
Characteristics of an RH panel
• The complete donor genome is represented
multiple times in each RH panel
• Each hamster cells has numerous fragments
of human DNA (~~~ 5-10 MB).
• Radiation Hybrid Mapping Information
Page
Ordering/Mapping STSs
• 1) PCR amplify STS in each hybrid (fusion
cell) in the RH panel. The results is scored
as either + (STS present) or – (STS absent).
• 2) Data is entered into a central database
and pattern matching software generates
spatial relationship.
Ordering/Mapping STSs
• Two markers (STS or other) that physically
lie near each other will show similar
patterns of retention or loss.
• New STSs are mapped by comparing the
pattern of positives and negatives with the
patterns in the database.
Assigning Chromosomal
Location
• Chr7: Radiation Hybrid Map
Expressed Sequence Tags (ESTs)
As of Dec. 2001, the ~10 million EST
records comprised ~72% of the
sequences in GenBank. ESTs from
mouse and human genomes total over
6 million. Although all of the original
ESTs were of human origin, NCBI’s
EST database (dbEST) now contains
ESTs from over 250 organisms.
What is an EST?
Short DNA sequence representing a gene
expressed in a particular tissue. A given
EST often represents a fraction of the gene.
Question- How do you know that the DNA
sequence is an expressed sequence?
Generation of an EST
• Generation of an EST is initiated by isolation of
mRNA from a tissue of interest.
• The mRNA is converted to a double-stranded
cDNA (complementary DNA) by the viral
enzyme reverse transcriptase.
• Both ends of the cDNA (usually) are sequenced.
These short sequences are the 5’ EST and the 3’
ESTs!
Human ESTs:
• There are ~ 4 million human
ESTs in GenBank . . . . . .
What is the value of ESTs?
• Rapid identification of genes.
Feb. 1992- Craig Venter and 14 co-workers
published the partial DNA sequence of of
2,375 genes expressed in the human brain.
This represented about half of the total
human genes known at the time.
How to sequence a
genome???
• 1) Quickly- focus on the genes and
their regulatory regions and human
polymorphisms.
• 2) Thoroughly and completely- every
nucleotide with 99.99% accuracy.
Big Deal?
• Venter and co-workers found novel
human genes that show strong
sequence similarities to interesting
genes from other species.
• Fact- Researchers hunting for a novel
gene are much more likely to find it in
dbEST than in the rest of GenBank.
Patenting of partial gene
sequences??
• NIH (Venter’s employer) applied for patents
on approximately 7,000 partial gene
sequences.
• Axel Kahn- “I compare this information to
the discovery of celestial galaxies. I would
patent the moon!”
‘Transcript Map’
• STSs derived from known genes and ESTs
 ‘Transcript Map’.
• (The assignment of expressed sequences to
specific chromosome regions is called
transcriptional mapping.)
GeneMap’99
• NCBI description:
- physical map of >35,000 human gene-based
markers, constructed by the International Radiation
Hybrid Mapping Consortium using a consistent set
of RH reagents and methodologies. Provides a
framework for accelerated sequencing efforts by
highlighting key landmarks (gene-rich regions) of
the chromosomes, and represents the cooperative
efforts of more than one hundred scientists
throughout the world.
What is an ‘Integrated Map’?
Map Integration
• Map intergration is cross-referencing
between various types of maps.
• This process in more difficult than it sounds
as the units of distance are different and do
not exactly translate (1 cR3000 = ~ 100 kb).
• GeneMap'99
The Genome Database (GDB) is the official repository
for genomic mapping data created by the Human
Genome Project.
• GDB stores and curates data generated by researchers engaged in
the mapping effort of the HGP. At present, GDB comprises
descriptions of the following types of objects:
• Regions of the human genome, including genes, clones, amplimers
(PCR markers), breakpoints, cytogenetic markers, fragile sites,
ESTs, syndromic regions, contigs and repeats.
• Maps of the human genome, including cytogenetic maps, linkage
maps, radiation hybrid maps, content contig maps, and integrated
maps. These maps can be displayed graphically via the Web.
•
• Variations within the human genome including mutations and
polymorphisms, plus allele frequency data.
• The Genome Database
SNPs = single nucleotide
polymorphisms
• Estimated number- every 500 or 1,000
nucleotides. Generally thought to be
biallelic.
• (mutation vs. polyporphism?)
Finding Disease Genes
• deCODE genetics
• Why Iceland?
Searching for Disease Genes almost
always start with the DNA of affected
individuals.
Process at Decode Genetics:
• 1) Identify people with a particular disease
• 2) Find affected people who are related in such a
way that they are likely to share genes ( pedigree)
• 3) Extract the DNA of these individuals
• 4) PCR amplify (robotically) SNPs along each
person’s chromosomes
• 5) Look for clusters of SNPs among the DNA of
patients from a single family.
• 6) Such clusters suggest  ?
Such clusters suggest a gene
involved in the disease is located
nearby.
• Big deal?
• If this data is correct, the gene has now been
linked to a particular chromosomal region.
Presumably the gene will soon be found.
• Where do you go next??
• Sequenom: From Code to Cure
• (see about us)
• Sequenom's scientists are interested in
changes in the frequency of SNPs as the
population ages. "We take advantage of the
fact that most human diseases are late-onset.
Age is a major risk factor . . .”
“If young people are carrying a
harmful variation, they're still well,
whereas an old person carrying that
same variation has a very high
chance that he's been made sick or
killed by it. You make the prediction
that variations that are harmful to
health should decline in frequency as
a function of age in the healthy
population.”
Charles Cantor (C.E.O. Sequenom):
• -Drugs target proteins
• ~500 known target found in the last century
• Next 2 years- ~ all impt. targets identified
Charles Cantor (C.E.O. Sequenom):
• Needed:
• a) markers [lots]
• b) populations [big]
• c) accurate precise tools [~30% of SNPs
are real]
Charles Cantor (C.E.O. Sequenom):
• Markers- SNPs- (Sequenom has 400,000 SNP
assays that are working).
• Population- Sequenom has recruited 15,000 blood
donors
• Assay- MALDI-TOF (Matrix-Assisted Laser
Desorption Ionization – Time of Flight).
Derivative of Mass Spec. Automated, high
throughput, accurate.
Charles Cantor (C.E.O. Sequenom):
• PCR assay involves 3 ddNTPs, and 1
dNTPs:
• _|||||||||||||||||||||||||||||||||____
A
• _|||||||||||||||||||||||||||||||||____
G
Charles Cantor (C.E.O. Sequenom):
• Identified 81 target genes*.
* Initially the disease associated with the
target gene is unknown.
Next Step- Twin Studies
Charles Cantor (C.E.O. Sequenom):
• Results  testing??
Result of test- AA, AG, or GG (crystal clear)
Implications of the test- unclear in many
cases.
Contrast HD testing with HIVR testing
Charles Cantor (C.E.O. Sequenom):
• treatment?
• Pharmacogenomics?? :
• Pharmacogenomics is the study of how an
individual's genetic inheritance affects the
body's response to drugs.
Jobs at Sequenom?
• Sequenom: From Code to Cure
The End