some techniques which look at more than one rna at a time

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of copies of protein? Function of protein?
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Problem - With Most Molecular Techniques Can Only
Work with ONE RNA at a Time -RT-PCR -Northern
Blots -Nuclease Protection
Some Techniques which look at more than one RNA
at a Time -Subtractive Hybridization -Differentia
l Display Problem Still need to isolate the
genes and figure out what you have after you
find changes in gene expression
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Overview of Microarray Technology First Step
The Genome Projects - Finding out the
Sequences Of all sorts of cDNAs and processing
them. Second Step Print Array of cDNAs, ESTs,
Oligos On Glass Slides - 2 cm x 2 cm - 6,400
genes/square!!!!!
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An Overview of Array Technology
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Step 6 Analysis by Computer
- DNA from the Frontal Cortex From Alcoholics
(Green) -DNA from the Frontal Cortex
of Non-Alcoholics - control (Red)
http//www.the-scientist.com/yr2001/feb/research_0
10205.html
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- 4 of 4,000 RNAs Tested Were Different Between
Alcoholics And Non-Alcoholics by 40 or More
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Two Methods of Printing Arrays
1. Spotting 1 kB cDNA amplified by PCR Spotted
onto coated glass slide
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2 -- Direct Oligonucleotide Addition - Uses
Chemical Blocking
GATAT ACGTC TTGGC
TTGG
TT
1. Tens of thousands of the same oligonucleotide
are made within a small square Area 2.
Oligonucleotides range in size from 8 to 70
nucleotides
lthttp//www.affymetrix.com/technology/synthesis.ht
mlgt
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Hybridization Must be Controlled - Data Must Be
Repeated -False Hybridization -Probes May Not
Bind Correctly ( a lot of controls need to
be Done)
Disadvantage - Only Relative Amounts of Activity
Can Be Seen. Go Back To Northern - RT- PCR to
See Actual Changes.
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Examples of Micro Array Analysis
- Brain Disease/Neurodegenerative
Disease -Cancer
-Malaria -- Parasitic Expressions - study changes
that happen in parasite Over the Course of
Disease
-Yeast Studies - First Microarray Studies in
Yeast -To Study Changes Between Aerobic and
Anarobic Bacteria
- Development - Studies Ongoing in Many
Organisms including Arabidopsis, Drosophila,
Yeast, and Rat
- SNP analysis to detect changes in cancer
causing genes, HIV protein
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Whats NEXT - PROTEIN MICROARRAYS - already
Developed although less widely used than
DNA Arrays - Spot Protein onto array -
Protein- Protein Interactions - Enzyme-Substrate
Interactions (used to detect kinases) - Small
Molecule Protein Interactions (receptor
binding) glutamate to receptor... -Protein -
Antibody Interactions
Howard Hughes Medical Institute
investigator Stuart L. Schreiber
and Gavin MacBeath