Ch 21 Genomics II Flashcards
Bioinformatics
the use of computer, math tools, and statistical analysis to analyze biological information
What is a microarray
Also reffered to as GENE CHIP.
makes monitoring of thousands of genes at one time possible
What is is: small silica, galss, or plastic slide taht is dotted with many different sequences of DNA, each correspoinding to a short sequence within a known gene
How are microarrays made?
1) spotting different samples of DNA onto a slide
2) Oglinucleotides directly synthesized on the surface of the slide
How to utilize a microarray?
1) Gather a mix of 3 mRNA
2) Add reverse transcriptase and poly dT primers and flourescent nucleotides
3) one strand cDNA created that is flourescently labeled
4) hybridize DNA into the microarray
5) view with Laser Scanner
* lit regions show complementary segements to the original mRNA
What are the applications of Microarraying?
1) cell specific gene expression
2) gene regulation
3) Elucidation of metabolic pathways
4) tumor proffiling
5) genetic variation
6) microbial strain indentification
7) DNA-protein binding
What is a diauxic shift?
The change from gycloysis to the TCA (tricarboxylic acid) cycle. Involves major changes in genes being expressed in carboxylate metabolism
How does yeast cell glycolisis work
When glucose available, metabolize glucose to smaller products. If oxygen present, breaks down further with TCA cycle
How does carbohydrate metabolism experiment work?
Measuring genes being expresss/reppresed during shift from glycolis to TCA cycle.
1) Put yeast in medium with 2% glucose. Let grow 21 hours.
2) At 9 hours take some cells out and then more cells every two hours after that.
3) Isolate mRNA (High salt binds low salt is released)
4) Add reverse transcriptase, poly DT, and flourescence.
* cDNA from 9 hours will be green and older will be red
5) mix red and green and hybridize into microarray
6) Analyze ratio of red: green and different times
Analyzing results via cluster analysis
identifying genes whose pattern of expression strongly correlates with one another.
CHIP *Chromatin immunopreciptiation
Can show if a protein can bind to a certian region of DNA in chromatin of LIVING cells
How does CHIP work?
1) find protein of interest
2) add formaldehyde which cross links DNA and Protein
3) lyse cell and sonicate DNA into small pieces
4) Add antibodies bound to beads that recognize protein of interest
5) centrifuge and take complexes from pellet (add chemical that breaks crosslinks)
6) Either conduct PCR using regions specific DNA OR covalently attach DNA linkers to ends of the DNA
7) If PCR amplified the protein was bound OR now conduct PCR using primers com. to linkers and flour. labled nucleotides.
8) Decature DNA and hybridize to microarray.
Proteome bigger than genome due to?
1) alternative splicing
2) RNA editing
3) Post-translational covalent modifications
Two Dimensional Gel Electrophoresis
Can seperate hundreds to thousands of proteins within a cell extract
1) Lyse sample of cells and load onto an isoelectric focusing gel
2) Lay tube gel onto SDS gel and seperate proteins according molecular weight.
What is an isoelectric focusing gel?
It is a gell in which proteins stop at zero charge location so the charge of proteins can be determined
What is antibody microarray and how does it work?
1) Antibodies spotted on microarray
2) labeled cellular proteins exposed to microarray
3) Cellular proteins bound to antibodies
* gives you antibodies that can target specific Proteins later on
