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 # years it would take to a chromosome location sequence the human genome 1967 none sequencing not possible yet genes mapped 4,000,000 years to finish at 1977 rate genes mapped 1000 years to finish at 1987 rate ,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 PageRadiation 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 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 cR 3000 = ~ 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.Regions of the human genome 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.Maps of the human genome Variations within the human genome including mutations and polymorphisms, plus allele frequency data.Variations within the human genome

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)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 HIV R 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