Final

1
(No Transcript)
2
How are we different?at the DNA level.
3
Methods of Detection

• Mendelian Genetics,
• Direct DNA Sequencing,
• RFLP analysis,
• Restriction Fragment Length Polymorphism,
• Allele Specific Oligonucleotides,
• DNA Microarrays

4
DNA Polymorphisms

• a DNA locus that has two or more sequence
  variations, each present at a frequency of 1 or
  more in a population,
• 1 in 700 frequency common in most species,
• less than 1 million loci in humans (1 in 3,000).
• five classes of polymorphisms.

5
Polymorphisms

• Single Base Pair Differences,
• Single Nucleotide Polymorphisms (SNPs),
• Microsatellites (short sequence repeats),
• Minisatellites (long sequence repeats),
• Deletions,
• Duplications.

6
RFLP AnalysisRestriction Fragment Length
Polymorphism

• Variations in the length of DNA fragments
  generated by restriction enzymes.
• the variations are caused by mutations that
  abolish or create cutting sites,
• RFLPs are inherited in a co-dominant fashion and
  can be used as genetic markers.

7
(No Transcript)
8
Single Nucleotide PolymorphismsSNPs
Eco RI site
SNPs, 98 of all polymorphisms.
9
Allele-Specific OligonucleotideASO

• short synthetic DNA probes able to differentiate
  between sequences differing by as little as 1
  base pair,

--AGTAGCTGTAGCT-- --TCATCGACATCGA--
Probe with fluorescent or radioactive dCTPs.
10
ASO and PCR

• subject gene is amplified using PCR,
• bound to a solid substrate,
• probed with labeled ASOs.

11
ASO
12
DNA Arrays

• DNA systematically arrayed at high density,
• comparative genomics,
• DNA hybridization to DNA,
• inter- and intra-species comparisons, etc.
• virtual genomes for expression studies,
• mRNA hybridization to DNA (cDNA) for expression
  studies,
• potential yet to be developed.

13
DNA Array
14
Probes/Targets

• ...Probes are the tethered nucleic acids with
  known sequence (ASO or cDNA),
• the DNA on the array,
• ...Target is the free nucleic acid sample whose
  identity/abundance is being detected,
• the labeled nucleic acid that is washed over the
  chip.

15
DNA-Probes

• cDNA arrays, DNA arrays,
• DNA Microarrays,
• oligonucleotide arrays,
• 2. DNA chips.

16
cDNA Microarrays

• ...denatured, double stranded probe DNA (500 -
  5000 bp) is dotted, or sprayed on a glass or
  nylon substrate,
• ...up to tens of thousands of spots per array,

17
DNA Chips

• oligonucleotides systematically synthesized in
  situ at high density (probes).

Affymetrix DNA Chip
18
Ordered Array of ASOs
...over a million different ASOs and controls can
be gridded per cm2.
19
Photolithography

• the process of using an optical image and a
  photosensitive substrate to produce a pattern,

• oligonucleotide synthesis can be inhibited by a
  protection group molecule,
• the protection group can be linked by a
  photosensitive bond, and thus cleaved by light.

20
(No Transcript)
21
(No Transcript)
22
Targets

• ...fluorescent targets,
• genomic DNA,
• cDNA, mRNA or cRNA for expression studies,

targets are washed over the chip for
hybridization.
23
Hybridization Detectionlabeled target is
hybridized to the array, unbound target is washed
away.

• fluorescent images are read by an optical
  scanner, and intensities are compared using
  algorithms to differentiate artifacts.

24
Screening for Genetic Disease

• Cystic fibrosis 75 of mutations are at the
  D508 deletion site,
• 8 are in three additional specific locations in
  the gene, the rest are spread across the length
  of the gene,
• Pre-Array tests yielded only an 83 chance of
  detecting a mutation.

25
Cystic fibrosis Detection

• Create a DNA chip with ASOs for wild-type Cystic
  fibrosis gene,
• approximately 4.5 kb of the 250 kb gene codes for
  the structural portion of the gene ( splice
  sites),
• 225 20-mers span 4.5 kb,
• 20 overlaps per 20-mer requires 4500 ASOs
  (grids), plus controls (2-3 per grid).

26
Creating the Mask

• computer algorithms are used to design the mask,
• creation of mask is now the limiting process,
  requires months to accomplish, and about 100,000
  per mask,
• masks have limited lifetimes, each array costs
  about 100 currently.

27
Cystic fibrosis Chip

• using photolithography, create a chip with ASOs
  to identify any difference from wild-type DNA,
• match results with mutations at known
  deleterious loci,
• catalog new deleterious loci.

28
1 Gene of Many

• with controls, the Cystic fibrosis gene may
  require up to 20,000 grids,
• new chips can accommodate up to 1 million grids,
• can look at 50 similarly sized genes on one
  chip.

29
4000 Genetic Diseases

• as genes are linked to diseases, quick,
  inexpensive tests can be performed to determine
  who carries specific mutations,
• gene must be mapped, cloned and sequenced,
• DNA chips designed, and data storage and analysis
  systems established.

30
gt906,600 SNPs arrayed
31
Genome Profiling

• with 906,000 SNPs and 946,000 additional genetic
  markers to measure copy gene number, genetic
  profiles can be made,
• choose SNPs in or near genes involved in traits
  or diseases,
• remember, linked genes are likely to travel
  together,
• or better, if the SNP is in an allele of
  interest, it can be readily identified,
• compare profiles over large populations.

32
(No Transcript)
33
How are we different?at the RNA level.
34
Northern Analysis

• DNA hybridizing to RNA,

35
DNA Arrays and Expression

• grid gene-specific ASOs onto the DNA chip, or
  known cDNAs onto microarrays,
• extract mRNA from a specific tissue,
• make fresh cDNA from the new mRNA,
• bind to the array for display.

36
http//www.bio.davidson.edu/courses/genomics/chip/
chip.html
37
Genes and Targets

• With many genome projects finished done, most, if
  not all of the genes identified can be gridded,
• presently, several completely sequenced genomes
  have been gridded,
• H. sapiens (gt20,000 genes)
• Arabidopsis (gt26,000 genes),
• C. elegans (gt22,500 genes),
• Drosophila (gt13,500 genes),
• yeast (gt6000 genes),
• more,
• drug identification, fundamental research, etc.

www.affymetrix.com
38
Gene Expression Technologies

• General Scheme Extract mRNA, synthesize labeled
  cDNA, Hybridize with DNA on the array,
• DNA Chips (Affymetrix) and MicroArrays can
  measure mRNA concentration of thousands of genes
  simultaneously,
• look for genes that are expressed similarly
  (clustering).

39
(No Transcript)
40
Gene/Drug Discovery

• genes involved in cancer and other diseases have
  been identified through a variety of techniques,
• genome expression analysis provides a means of
  discovering other genes that are concomitantly
  expressed,
• genome expression analysis provides a means of
  monitoring drug/treatment regimes.

41
(No Transcript)
42
Applications

• Can study the role of more than 1700 cancer
  related genes in association with the (rest) of
  the genome,
• Define interactions and describe pathways,
• Measure drug response,
• Build databases for use in molecular tumor
  classifications,
• benign vs. cancerous, slow vs. aggressive.

43
Extended Applications

• Water quality testing (4 hours vs. 4 days),
• Environmental watchdogs,
• Fundamental research on non-human subjects,
• Direct sequencing of related species for
  evolutionary studies,
• Comparisons of gene regulation between closely
  related species,
• etc.

44

• Human and chimp DNA is 98.7 similar,
• But, we differ in many and profound ways,
• Can this difference be attributed, at least in
  part, to differences in gene expression, rather
  than differences in the actual gene and gene
  products?

Monday