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 templatePrimers (anneal to flanking sequences)DNA polymerasedNTPsMg2+

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 Primersw/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)

African-American Asian-Chinese Hispanic-Mexican Caucasian Caucasian

Whose genome did Celera sequence?Supposedly:

Actually:

Celera’s genome is Craig Venter’sScience v. 291, pp 1304-1351

Cloning Vectors

• Plasmid• Phagemid• Cosmid, fosmid• YACs• BACs• PACs

BAC Construction Scheme

T7 Insert SP6

Insert size ; 50-300Kb ( mean 110kb)

BAC vector (pIndigo 536)

Application Of BAC library

BAC library

Whole genome sequencing

Construction of integrated ge-netic and physi-cal map

Fine mapping for interest gene

Chromosome walking & con-tig assembly

Positional cloning

Fluorescent in situ hybridization

“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 double-stranded 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:

4h 8h 12h

1d15’

30’

1h 2hNA4h 8h 12h

1d15’

30’

1h 2hNA

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