positional cloning animation

Genomics

What its genomics?

Genomics

Structural Genomics

Positional cloning

Functional Genomics

Functional Genomics Techniques

How can we measure the pattern of gene expression in a given tissues over time?

Northern blots In situ hybridization

Microarrays and Microchips

Allow you look at expression of all the potential mRNAs in a cell at the same time

DNA microarrays

•DNAs spotted on slide•025-1 nl•Spots 100 µm diameter•200-150 µm apart

Spots DNAs directly on chip!

Synthesize oligonucleotides on surface of a chip

Photolithography

Growing oligonucleptides on a glass surface

Oligonucleotide whole genome arrays

Can use genome sequences to generate oligonucleotide arrays to identify each gene product in a genome.

How long must an oligonucleotide be to uniquely identify one human gene?

Sequence of n bases will occur every 4n bases

What is the minimum number of bases that will occur about once in the human genome

4n bases such that n results in 4n at least the size of human genome

4n = 3.5 X 109

n = 16

Affymetrix Inc.

Check out this animation

http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

Statistics 6116 Yeast Genes96 Intergenic regions+ lots of control samples

Dr. Patrick O. Brown Stanford

Positional Cloning

A strategy for cloning genes based solely on map position

Cloning Disease Genes

Requires knowledge of the protein

Sickle cell anemia

Huntingtons Chorea

Cystic Fibrosis

Map based cloning

Involves isolating a marker piece of DNA whose location is known and which is tightly-linked to the gene you are trying to clone

Map based cloningNorth AmericaUnited StatesCascade RangeMount RanierPuget SoundDowntown SeattleSeattle CenterSpace Needle

Cystic Fibrosis

First human disease gene cloned solely on the basis of position determined by linkage analysis

Took 4 years Lap-Chee Tsui, Francis Collins and John Riordan isolated the gene in 1989

StrategyScreened a large number of families for RFLP linked to CFFound RFLP called DOCR-917 that was tightly linked to CFSomatic cell hybrids showed that DOCR-917 is on Ch. 71985: reported linkage between 7q22 and 7q31.1 (Met andD788)– 1.7 Mb apart

How do you know when you have arrived?

Human genes have CpG islands at 5’ endsImportant genes are often highly conserved

(zoo blots)

Gene should be transcribedGene sequence should be different in normal and diseased individualsCF gene (CFTR) finally isolated from cultured sweat gland cells

Cystic Fibrosis GeneUsed probe to isolate cDNA (6000 bp) from diseased and normal individualsDiseased gene had deletion of 3 bpNormal ATC ATC TTT GGT (Ile Ile Phe Gly)Disease D508 ATC AT T GGT (Ile Ile Gly)68% of all CF caused by D508Remaining 32% caused by > 190 different mutations