DNA microarrays allow scientists to analyze thousands of genes simultaneously. They work by attaching DNA probes to a plate to measure gene expression levels in different samples. The process involves isolating mRNA from samples, converting it to labeled cDNA, hybridizing it to the microarray plate, and scanning the plate to analyze gene expression differences between samples. Microarrays have benefits like speed and analyzing many genes at once, though they also produce large amounts of data to analyze. Future uses include disease diagnosis, pharmacogenomics, and toxicogenomics research.

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.