Genomics What its genomics?
Genomics Structural Genomics
Functional Genomics Positional cloning
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 Genes 96 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 tightlylinked to the gene you are trying to clone
Map based cloning
North America United States Cascade Range Mount Ranier Puget Sound Downtown Seattle Seattle Center Space 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
Strategy
Screened a large number of families for RFLP linked to CF Found RFLP called DOCR-917 that was tightly linked to CF Somatic cell hybrids showed that DOCR-917 is on Ch. 7 1985: 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’ ends Important genes are often highly conserved
(zoo blots)
Gene should be transcribed Gene sequence should be different in normal and diseased individuals CF gene (CFTR) finally isolated from cultured sweat gland cells
Cystic Fibrosis Gene
Used probe to isolate cDNA (6000 bp) from diseased and normal individuals Diseased gene had deletion of 3 bp Normal ATC ATC TTT GGT (Ile Ile Phe Gly) Disease D508 ATC AT T GGT (Ile Ile Gly) 68% of all CF caused by D508 Remaining 32% caused by > 190 different mutations