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 sequencing, 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
Cloning Vectors • Plasmid • Phagemid • Cosmid, fosmid • YACs • BACs • PACs
Application Of BAC library Whole genome sequencing Chromosome walking & contig assembly Fluorescent in situ hybridization BAC library Fine mapping for interest gene Construction of integrated genetic and physical map Positional cloning
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
P. trifoliata C. paradisi 4h 8h 12h 1d 4h 8h 12h 1d NA 15’ 30’ 1h 2h NA 15’ 30’ 1h 2h CORc115 PtCBF
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
Sequence Conservation • Homology searches
Gene Complementation • Mutants • Overexpression • “Knockouts” • RNAi • Reporter assays