1. PHYSICAL MAPPING AND POSITIONAL CLONING

2. Linkage mapping – Flanking markers identified – 1cM, for example Probably ~ 1 MB or more in humans Need very many families to get closer than this in human, or very large populations

5. What to Do Next? Identify genes in this region Then determine what is the gene of interest

6. What to do next? In 1990, clone region around markers, make physical map(s), look for genes experimentally In 2000, use better physical maps, at least in some organisms In 2010, use sequencing, bioinformatic knowledge, experimental proof still necessary

7. Physical Mapping A physical map is primarily based on the locations of landmarks along a DNA molecule and units of distance are expressed in base pairs.

8. Low Resolution Physical Mapping Cytogenetic map In situ hybridization

9. Chromosome Sorting Flow cytometry Used in library construction Also for chromosome paints

13. Somatic Cell Genetics in Mammals

16. Radiation Hybrid Mapping

18. Top-down mapping

21. Bottom-up Mapping (Contig Maps, Mapping With Ordered Clones)

23. Cloning Vectors Plasmid Phagemid Cosmid, fosmid YACs BACs PACs

27. Application Of BAC library BAC library Whole genome sequencing Construction of integrated genetic and physical map Fine mapping for interest gene Chromosome walking & contig assembly Positional cloning Fluorescent in situ hybridization

28. Maximum pyrosequencing read lengths currently are 300-500 nt. Commercial applications: 454 Life Sciences Genome Sequencer FLX Generate 400 million nt in 10 hours $5-7K USD per run $1M for mammalian genome

32. Zoo Blot

34. CpG islands

35. Northern Blots

36. 4h8h12h1d 15’30’1h2hNA4h8h12h1d 15’30’1h2hNA P. trifoliataC. paradisi PtCBF CORc115

37. Real-time Quantitative PCR: Measures the abundance of DNA as it is amplified. Useful for quantitatively measuring the levels of mRNA in a sample. Uses reverse transcriptase to generate cDNA for the template. Can also be used to quantitatively estimate fraction of DNA from various organisms in a heterogenous sample (e.g, can be used to measure abundance of different microbes in soil sample). Fluorescent dye, SYBR Green, is incorporated into PCR reaction. SYBR Green fluoresces strongly when bound to DNA, but emits little fluorescence when not bound to DNA. SYBR Green fluorescence is proportional to the amount of DNA amplified, detected with a laser or other device. Experimental samples are compared to control sample with known concentration of cDNA.

38. Fig. 10.9 SYBR Green binds to double-stranded DNA and fluoresces

39. Sequence Conservation Homology searches

40. Array Technology

43. Gene Complementation Mutants Overexpression “Knockouts” RNAi Reporter assays