DNA microarray.ppt

DNA Microarray

1. DNAMICROARRAY
S.KOUSALYA M.Sc., M.Phil
Assistant Professor
Department of Microbiology
VIAAS
DEPARTMENT OF MICROBIOLOGY
VIVEKANANDA ARTS AND SCIENCE COLLEGE FOR
WOMEN SANKAGIRI

2. What is DNA Microarray?
DNA microarray have been developed as a
method for rapidly analysing the expression of
all genes.

3. Perform the microarrray
currently different technologies were
used. Most commonly used two system.
1) Affymetrix chips (ssDNA oligo)
2) Stanford chips (PCR)
The PCR product spotted to specific
location.
DNA dried heat 100 C for 2 sec, fixed UV
cross linking and to make ssDNA.

4. The Plate.
Usually made commercially.
Made of glass, silicon, or nylon.
Each plate contains thousands of spots, and each
spot contains a probe for a different gene.
A probe can be a ssDNA or a synthetic
oligonucleotide.
Probes can either be attached by robotic means,
where a needle applies the ssDNA to the plate,
or by a method similar to making silicon chips or
affymetrix means, where using synthetic
oligonucleotide.

5. cDNA
The cDNA contain 3 normal deoxynucleotide
triphosphate & 1 is fluorescently labelled one.
1. Green colour (Cy3) indicate expressed gene.
2. Red colour(Cy5) indicate not expressed.
Each spot monitored using flurescent
confocal microscope.

6. STEP 1: Isolate mRNA.
Extract the RNA from the samples.
After isolating RNA, the mRNA will be
extracted.

7. STEP 2: Create LabelledDNA.
Add a labellingmix to the RNA.
The labellingmix contains poly-T
(oligo dT) primers, reverse
transcriptase (to make cDNA),
and fluorescently dyed
nucleotides.
We will add cyanine 3 (fluoresces
green) to the healthy cells and
cyanine 5 (fluoresces red) to the
cancerous cells.
The primer and RT bind to the
mRNA first, then add the
fluorescently dyed nucleotides,
creating a complementary strand
of DNA

8. STEP 3: Hybridization.
Apply the cDNA we
have just created to a
microarray plate.
When comparing two
samples, apply both
samples to the same
plate.
The ssDNA will bind to
the cDNA already
present on the plate.

9. STEP 4: Microarray Scanner.
The laser causes the hybrid
bonds to fluoresce.
The camera records the images
produced when the laser scans
the plate.
The computer allows us to
immediately view our results
and it also stores our data.

10. STEP 5: Analyze the Data.
GREEN–the healthy
sample hybridized
(expressed)
RED–the
diseased/cancerous sample
hybridized (not expressed)
YELLOW-both samples
hybridized equally to the
target DNA.

11. Problems.
Oligonucleotide –Some time
contamination will occur.
DNA Microarray only detects
whether a gene is expressed or
not.
Continuous/large amounts of
data was given not for brief.
http://www.stuffintheair.com/very-big-problem.html

12. The Future of DNA Microarray.
Gene discovery.
Disease diagnosis : Classify the types of cancer
on the basis of the patterns of gene activity in
the tumor cells.
Pharmaceuticals :The study of drug and how to
response the patients.
Toxins : Microarray technology allows us to
research the impact of toxins on cells.

13. THANK YOU