positional cloning animation
TRANSCRIPT
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