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PHYSICAL MAPPING AND POSITIONAL CLONING • 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 What to Do Next? • Identify genes in this region • Then determine what is the gene of interest 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 bioinformatic knowledge, experimental proof still necessary 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. Low Resolution Physical Mapping • Cytogenetic map • In situ hybridization Chromosome Sorting • Flow cytometry • Used in library construction • Also for chromosome paints Pulsed Field Gel Electrophoresis Polymerase Chain Reaction (PCR) Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning). Cloning DNA is time consuming and expensive (>>$15/sample). Probing libraries can be like hunting for a needle in a haystack. PCR, “discovered” in 1983 by Kary Mullis, enables the amplification (or duplication) of millions of copies of any DNA sequence with known flanking sequences. Requires only simple, inexpensive ingredients and a couple hours. DNA template Primers (anneal to flanking sequences) DNA polymerase dNTPs Mg2+ Buffer Can be performed by hand or in a machine called a thermal cycler. 1993: Nobel Prize for Chemistry Fig. 9.3 Denature Anneal PCR Primers Extend PCR Primers w/Taq Repeat… Somatic Cell Genetics in Mammals Radiation Hybrid Mapping Fluorescent Dye Dideoxy-sequencing Top-down mapping Bottom-up Mapping (Contig Maps, Mapping With Ordered Clones) Whose genome did Celera sequence? Supposedly: African-American Asian-Chinese Hispanic-Mexican Caucasian Caucasian Actually: Celera’s genome is Craig Venter’s Science v. 291, pp 1304-1351 Cloning Vectors • • • • • • Plasmid Phagemid Cosmid, fosmid YACs BACs PACs BAC Construction Scheme BAC vector (pIndigo 536) T7 Insert Insert size ; 50-300Kb ( mean 110kb) SP6 Application Of BAC library Chromosome walking & contig assembly Whole genome sequencing Fluorescent in situ hybridization BAC library Fine mapping for interest gene Positional cloning Construction of integrated genetic and physical map “virtual autorad” - real-time DNA sequence output from ABI 377 1. Trace files (dye signals) are analyzed and bases called to create chromatograms. 2. Chromatograms from opposite strands are reconciled with software to create doublestranded sequence data. 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 Zoo Blot CpG islands Northern Blots 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. Fig. 10.9 SYBR Green binds to double-stranded DNA and fluoresces Real-time Quantitative PCR amplification plot: 1d 12h 8h 4h 2h 1h 30’ 15’ NA 1d 12h 8h 4h 2h 1h 30’ 15’ NA P. trifoliata C. paradisi PtCBF CORc115 Use trapped exons to identify candidate genes from cDNAs Sequence Conservation • Homology searches Array Technology Gene Complementation • • • • • Mutants Overexpression “Knockouts” RNAi Reporter assays