Download Arrays

The Human Genome Project and
~ 100 other genome projects:
• Done!!
• What do you find??
•  predictions of all genes/protein sequences.
What’s next??
Brief History of Molecular Genetics
• Pre-Genomic Era- Study of Individual Genes using
techniques of Molecular Biology.
• Genomic Era- Sequencing of entire genomes.
Analytical techniques include Sequencing and Gene
Finding.
The new way of doing biologyPost-Genomic Era
• Genome Analysis of Gene
Expression. Monitoring activity
(turn on, turn off) of all genes
together.
• #1 tool- DNA microarrays
Microarray Technology?
What is it??
The future . . .
• DNA microarrays (also
called DNA chips or Gene
chips) are miniaturized
laboratories for the study of
gene expression.
• Microarrays are small pieces of
glass (or silicon or nylon) about 2
or 3 cm square, coated with DNA.
• The DNA is spotted by high
speed robots in a very precise
pattern. The droplets of
DNA (measured in pL or nL)
are separated by _x_
microns.
• The surface of the array is covered
with thousands, tens of thousands, (or
soon with hundreds of thousands) of
spots, each spot containing a different
DNA oligomer.
• DNA Microarray Technique (animatedrequires Flash plug-in)
• Each oligomer in a DNA
microarray can serve as a probe
to detect a unique,
complementary DNA or RNA
molecule.
Many microarray experiments
have been performed with Yeast.
• Genome is completely sequenced and
well annotated.
• Select a PCR primer pair that amplifies
each ORF.
• My arrays- all yeast genes represented
(70mers).
PCR primers for sale . . .
• To our sales list we add Yeast ORF
specific primers for over 6,000 ORFs!
These primers have been designed to
amplify, from genomic DNA, the
complete coding region including the
start and stop codons. For yeast this is
possible as very few yeast genes
contain introns.
Common Protocol
In yeast (or other single-celled organisms)
compare cells grown in two different
conditions.
• Why? What information are biologists
searching for?
• GCAT Animation
See Microarray Data
• The Stanford Microarray Database microarray data storage, retrieval, analysis,
visualization, and database software
There are two distinct way of
making DNA arrays:
• DNA microarrays: Also referred to as
"microarrays." Non- porous solid supports,
such as glass have facilitated miniaturization
and fluorescence based detection. About
10,000 cDNAs can be robotically spotted onto
a microscope slide and hybridized with a
double labeled probe, using protocols
pioneered by Pat Brown and colleagues at
Stanford.
Gene Chips or DNA Chips
• Oligomers synthesized directly on the chips
(pioneered by Affymetrix). GeneChip ® is an
Affymetrix product where they have adapted
photolithographic masking techniques used in
semiconductor manufacture to produce arrays with
400,000 distinct oligonucleotides.
Affymetrix – Gene Chips
• DNA is short (25 nt)
• Small variances (SNPs) can be detected
• Only one Dye can be used at a time
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DNA Microarrays
cDNA is spotted on glass plates
cDNA is from 100nt to 2kb in length
Very efficient hybridization
Small variances (SNPs) can’t be seen
#1 use of Microarrays- Investigate
Changes in Gene Expression.
Compare cells before and after perturbation:
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Nutritional
Environmental
Disease
Toxin
Different Stages of Development
Human Cells
• Compare normal tissue vs. diseased tissue.
• DNA Microarray Technique (animatedrequires Flash plug-in)
• PNAS -- article
Researchers love DNA chips;
Doctors will soon love DNA chips
• Researchers love DNA chips because they
give a huge amount of information, fast, at
low cost.
• Doctors will soon learn to love them because
there are many times when a doctor would
like to know something about a patient's
genes (such as whether the patient is likely
to respond well to a certain drug). When the
price comes down enough, microarrays will
likely become routine tools in the doctor's
office.
Clinical Applications?
• If this research pays off, scientists and
doctors will see a new, more
informative face of cancer for individual
tumors. That could produce tremendous
gains in cancer care.
• Doctors could better match patients to
the best treatment. That could improve
the outcome and spare patients the side
effects of therapies that won't help.
• Science -- Putting Gene Arrays to the Test
• Science -- Revolution in Diagnostics
• Since the development of DNA
microarray technology in the
late 1990s, it has become
apparent that the increase in
available gene expression data
will eventually parallel the
growth of the sequence and
structure databases.