DNA microarrays allow researchers to analyze gene expression levels across thousands of genes simultaneously. A DNA microarray contains many DNA probes attached to a solid surface in a regular pattern. Researchers isolate mRNA from samples, convert it to cDNA, and label the cDNA with fluorescent dyes. They then hybridize the labeled cDNA to the probes on the microarray. A scanner detects the fluorescence at each probe location, allowing researchers to compare gene expression levels between samples by the intensity and color of fluorescence. Microarrays have applications in medicine, agriculture, forensics and toxicology by enabling the comparison of gene expression in different tissues or in response to different conditions.

1. DNA
MICROARRAY
Neeraj Sharma
MSc Medical Microbiology
2nd year

2. What we will be discussing…
• What is DNA microarray?
• The purpose of using DNA microarray.
• Principle
• Steps to perform a microarray.
• Types
• Applications

3. Introduction
• DNA chip or a Biochip.
• Collection of microscopic
DNA spots attached to solid
surface.
• The probe sequences are
designed and placed on an
array in a regular pattern of
spots.

4. • The chip or slide is usually made
of glass or nylon and is
manufactured using technologies
developed for silicon computer
chips.
• Each microarray chip is arranged
as checkerboard of 105 or 106
spots or features, each spot
containing millions of copies of a
unique DNA probe (often 25 nt
long).
• Each spot contains pico moles of
(10-12 moles) of specific DNA
sequence known as probe.

5. Purposes.
• So why do we use DNA microarray?
o 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.
o To observe genomic gains and losses. Microarray Comparative
Genomic Hybridization (CGH)
o To observe mutations in DNA.

6. Principle
• The core principle behind microarrays
is hybridization between two DNA
strands, the property of
complementary nucleic acid
sequences to specifically pair with
each other by forming hydrogen
bonds between
complementary nucleotide base pairs.
• A high number of complementary
base pairs in a nucleotide sequence
means tighter non-covalent bonding
between the two strands. After
washing off non-specific bonding
sequences, only strongly paired
strands will remain hybridized.

7. Steps
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.

9. 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.

10. STEP 3: Create Labelled
DNA.
 Add a labelling mix to the RNA.
The labelling 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

11. 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.

12. 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.

13. STEP 6: Analyze the Data.
 GREEN – the healthy sample hybridized more
than the diseased sample.
 RED – the diseased/cancerous sample
hybridized more than the non-diseased sample.
 YELLOW - both samples hybridized equally
to the target DNA.
 BLACK - areas where neither sample
hybridized to the target DNA.
 By comparing the differences in gene
expression between the two samples, we can
understand more about the genomics of a
disease.

15. Types
• Spotted
• DNA fragments (usually
created by PCR)or oligos are
stuck to glass slides
• The size of the fragment can
be any length (usually 500 bp-
1 kb)
• The size of the oligos range
from 20-100 nts
• These arrays can be created in
individual labs using
“affordable” equipment

16. • Affymetrix
• Affymetrix arrays are typically
limited to oligos of 20-25 nts
• The probes on these arrays are
synthesized using a light mask
technology
• Photo-sensitive reactions are
used to remove a blocking
group and then extend
• It is very costly to fabricate
masks for a new array design
• Not commonly used for
custom arrays

17. Applications
• Medical field : - Cause of disease
Disease identification
Disease treatment
• Agriculture
• Forensic
• Toxicology

18. • Detect the presence or absence of specific genes.
• Compare genes from two different sources.
• They can see how the external genes are affecting the external
stimuli.

19. Printing of the probes in microarray.

20. Thank You