How are we different? …at the DNA level.

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 hundreds frequency common in most species, • about 10 million loci in humans (1 in 3,000). • five classes of polymorphisms. …including humans,

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.

8. Single Nucleotide PolymorphismsSNPs Eco RI site …SNPs, 98% of all polymorphisms.

9. --AGTAGCTGTAGCT-- --TCATCGAaATCGA-- mismatch no binding 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. labeled wt, D508 Bind subject DNA (PCR product) to filter, ... probe with labeled ASOs, seperately. ASO Cystic fibrosis pedigree

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. solid substrate DNA 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. nucleic acid is spotted onto the substrate. nucleic acid is synthesized directly onto on the substrate. DNA-Probes • cDNA arrays, DNA arrays, • DNA Microarrays, • oligonucleotide arrays, 2. DNA chips.

16. quill technology... cDNA Microarrays ...denatured, double stranded probe cDNA (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. linker molecule 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.

22. Targets ...fluorescent targets, • genomic DNA, • cDNA, mRNA or cRNA for expression studies*, for future reference, …targets are washed over the chip for hybridization.

23. Hybridization Detection…labeled 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 known 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. Genotyping >906,600 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. decodeme 23andme consyl knowme

34. How are we different?…at the RNA level.

35. Northern Analysis DNA hybridizing to RNA,

36. 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.

37. http://www.bio.davidson.edu/courses/genomics/chip/chip.html

38. 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 (>20,000 genes) • Arabidopsis (>26,000 genes), • C. elegans (>22,500 genes), • Drosophila (>13,500 genes), • yeast (>6000 genes), • more, • drug identification, fundamental research, etc. www.affymetrix.com

39. 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).

41. 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.

43. 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.

44. Extended Applications • Water quality testing (4 hours vs. 4 days), • Environmental watchdogs, • Fundamental research on non-human subjects, • DNA sequence identification of related species for evolutionary studies, and organism identification, • Comparisons of gene regulation between closely related species, • etc.

45. Exam #2 Mean = 70.1% Median = 87.3% Mode = 70.0% A range: | | B range: | | | | | | | C range: | | | | | | | D range: | | | | | | | | Failing: | | | | | |

46. 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