it tells the basics of bio informatics

2. DNA Microarray.
By- Jain Hemant

3. What is DNA Microarray?
• Scientists used to
be able to
perform genetic
analyses of a few
genes at once.
DNA microarray
allows us to
analyze thousands
of genes in one
experiment!

4. Purposes.
• So why do we use DNA
microarray?
– To measure changes in
gene expression levels –
two samples’ gene
expression can be
compared from different
samples, such as from
cells of different stages of
mitosis.
– To observe genomic gains
and losses. Microarray
Comparative Genomic
Hybridization (CGH)
– To observe mutations in
DNA.

5. 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.

6. • A probe can be a cDNA fragment or a
synthetic oligonucleotide, such as BAC
(bacterial artificial chromosome set).
• Probes can either be attached by robotic
means, where a needle applies the cDNA to
the plate, or by a method similar to making
silicon chips for computers. The latter is
called a Gene Chip.

7. Let’s perform a microarray!
1) Collect Samples.
2) Isolate mRNA.
3) Create Labelled DNA.
4) Hybridization.
5) Microarray Scanner.
6) Analyze Data.

8. STEP 1: Collect Samples.
 This can be
from a
variety of
organisms.
We’ll use two
samples –
cancerous
human skin
tissue &
healthy
human skin
tissue

10. STEP 2: Isolate mRNA.
• Extract the RNA from the samples. Using either a
column, or a solvent such as phenol-chloroform.
• After isolating the RNA, we need to isolate the mRNA
from the rRNA and tRNA. mRNA has a poly-A tail, so
we can use a column containing beads with poly-T
tails to bind the mRNA.
• Rinse with buffer to release the mRNA from the
beads. The buffer disrupts the pH, disrupting the
hybrid bonds.

11. STEP 3: Create Labelled DNA.
Add a labeling mix to the
RNA. The labeling mix
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

12. STEP 4: 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.

13. STEP 5: LASERS!

14. STEP 5: Microarray Scanner.
 The scanner has a laser, a
computer, and a camera.
 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.

15. Benefits.
• Relatively affordable (for some people!),
about $60,000 for an arrayer and scanner
setup.
• The plates are convenient to work with
because they are small.
• Fast - Thousands of genes can be analyzed at
once.

16. Problems.
• Oligonucleotide libraries –
redundancy and contamination.
• DNA Microarray only detects
whether a gene is turned on or
off.
• Massive amounts of data.
http://www.stuffintheair.com/very-big-problem.html

17. 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.
• Pharmacogenomics = is the study of correlations between
therapeutic responses to drugs and the genetic profiles of the
patients.
• Toxicogenomics – microarray technology allows us to
research the impact of toxins on cells. Some toxins can
change the genetic profiles of cells, which can be passed on to
cell progeny.