DNA

Gene A

Transcriptional ControlImprintingHistone Acetylation

# of copies of RNA?

Post TranscriptionalProcessingmRNA StabilityTranslational Control

Post Translational ControlProtein stability

# of copies of protein?Function of protein?

Problem - With Most Molecular TechniquesCan 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-Differential Display

Problem: Still need to isolate the genes and figure out what you have

after you find changes in gene expression

Overview of Microarray Technology

First Step: The Genome Projects - Finding out the SequencesOf 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!!!!!

An Overview of Array Technology

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- DNA from the Frontal CortexFrom Alcoholics (Green)

-DNA from the Frontal Cortex ofNon-Alcoholics - control (Red)

http://www.the-scientist.com/yr2001/feb/research_010205.html

Step 6: Analysis by Computer

- 4% of 4,000 RNAs Tested Were Different Between AlcoholicsAnd Non-Alcoholics by 40% or More

Two Methods of Printing Arrays

1. Spotting

1 kB cDNA amplified by PCRSpotted onto coated glass slide

#2 -- Direct Oligonucleotide Addition- Uses Chemical Blocking

<http://www.affymetrix.com/technology/synthesis.html>

TT TTGG

GATATACGTCTTGGC

1. Tens of thousands of the same oligonucleotide are made within a small square Area2. Oligonucleotides range in size from 8 to 70 nucleotides

Hybridization Must be Controlled- Data Must Be Repeated-False Hybridization-Probes May Not Bind Correctly ( a lot of controls need to beDone)

Disadvantage - Only Relative Amounts of Activity Can Be Seen.Go Back To Northern - RT- PCR… to See Actual Changes.

-Malaria -- Parasitic Expressions - study changes that happen in parasiteOver the Course of Disease

Examples of Micro Array Analysis

- Brain Disease/Neurodegenerative Disease

-Cancer

-Yeast Studies - First Microarray Studies in Yeast -To Study ChangesBetween 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

What’s NEXT - PROTEIN MICROARRAYS

- already Developed although less widely used than DNAArrays

- 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