DNA Microarray for gene expression applied in medical condition for comparision of gene expressed in infected individual to that of normal individual or healthy individual.

1. Bestowed by:- Abhay Pandit

2.  Also known as DNA arrays / DNA chips / biochips.
 DNA microarray is an orderly arrangement of thousands
of identified sequenced genes printed on an
impermeable solid support.
 Solid support may be of glass, silicon chips or nylon
membrane.
 Each identified sequenced gene on the support
corresponds to a fragment of genomic DNA, cDNA, PCR
products represents a single gene.
DNA microarray Technology?

3.  DNA microarray technology gave a
major breakthrough in experimental
molecular biology.
 First, article describing the
application of DNA microarray
technology was published in the
scientific literature by Patrick Brown
and his colleageus at Stanford
University.
 In 1995, Patrick Brown et. al.
measured the differential expression
of 45 Arabidopsis genes by means of
simultaneous two color flourescence
hybridisation.
History
Source:http://pgsb.helmholtzmuenchen.d
e/plant/static/images/athal_blumeninsch
waben.jpg
Arabidopsis thaliana

4. DNA Microarray
Glass DNA
Microarray
High-Density
Oligonucleotide
Microarray
Types of DNA Microarrays

5. Glass DNA Microarrays
Glass DNA microarray was the first type of DNA microarray
technology developed.
Pioneered by Patrick Brown and his colleagues at Stanford
University.
cDNA or oligonucleotide are applied to a glass microscope
slide by a robotic device and amplified by PCR, which
deposits nanoliter quantities of cDNA solution.
Glass slide usually have excellent chemical resistance against
solvents and with low intrinsic fluorescence properties.
Moderate sized glass cDNA microarray bears about 10,000
or more spots on an area of 3.6cm2.

6.  It is a sophisticated platform.
 First developed by Stephen Fodder et al.
(1991).
 Affymetrix Industry leader in the field of
microarray, manufactured “GeneChips”
which refers to its high density
oliginucleotide based DNA arrays.
 Made by synthesis of oligonucleotides
on the surface of the slide, not by
deposition of PCR products.
 Commerial version of Affymetrix gene
chips hold upto 5,00,00 probes/site in a
1.28cm2 chip area.
In situ oliginucleotide array format
Source:https://github.com/bioinformaticscoresharedtrainin
g/microarrayanalysis/blob/master/affymetrix.Rmd

7. Difference b/n Glass microarray &
DNA chip.

8.  Based on the fact that complementary sequences of DNA can be used
to hybridize immobilized DNA molecules.
 A microarray experiment involves the comparision of a query or
experimental sample representing the expression pattern of genes in a
specific set of conditions, with a control sample representing all the
genes that are expressed in the cells/tissues to be analysed.
 This involves 4-major stages;
I. Sample preparation and labelling.
II. Hybidization
III. Washing
IV. Image acquisition and data analysis.
Principle of DNA Microarray

9. Basic Flow Chart : Microarray
Collect mRNA
molecule from a
cell
Use Reverse
Transcriptase(RT)
enzyme to produce
cDNA molecules from
the mRNA
Label cDNA with
flourescent Dyes
Prepare microarray/DNA
chip(cDNA from
reference genes or
oligonucleotide
mixture)
Place labeled
cDNA on
microarray slide
Hybridization of
labeled cDNA with
cDNA(complemena
try) on microarray
Scan array slide.
More flourescence,
more intensity of
expression

10.  Sample preparation starts by isolating a total RNA
containing mRNA that ideally represents quantitative
copy of genes expressed at the time of sample
collection (i.e., experimental and reference sample).
Since, overall success of any microarray experiment
depends on the quality of the RNA.
 mRNA from sample are converted into cDNA by an
enzyme called reverse transcriptase.
 Control/Reference cDNA is labeled with a green
flourescent tag by including Cy3 Cyanine dye.
 Experimental/diseased cDNA is labeled with a red
flourescent tag by Cy5 Cyanine dye.
Sample preparation and
labelling

11.  Joining of two complementary strands of DNA to form a double-
stranded molecule.
 Complementary hybridization-
-CTAGCAGG - Actual gene
-GATCGTCC - cDNA (Reverse Transcription)
-CUAGCAGG - mRNA
 Here, the labelled cDNA (Sample and Control)are mixed together.
 Followed by purification to remove contaminants such as primers,
unincorporated nucleotide, proteins, lipids and carbohydrates.
 Purified mixture is competitively hybridised against denatured PCR
product or cDNA molecules spotted on a glass slide.
 Labelled cDNA will bind to its appropriate complementary target
sequence on the immobilised array.
Array Hybridization

12.  Before, hybridisation microarray slides are incubated at
high temperature with solutionsof saline-sodium buffer
(SSC), Sodium Dodecyl Sulfate(SDS) and BSA to reduce
background due to nonspecific binding.
i) to remove any labelled cDNA that did not hybridise on
the array.
ii) To remove stringency of the experiment to reduce
cross hybridisation.
Stringency can be achieved by either increasing
temperature or lowering the ionic strength of
buffer.
Hybridization contd…
Washing:-

13. Source:- https://basicmedicalkey.com/dna-microarray/

14. Final step of microarray experiments.
An image of the surface of hybridised array is produced.
Slide is dried and placed into a laser scanner to
determine how much labelled cDNa is bound to each
target spot.
Characteristic emmission spectra of target spot is
obtained by excitation with specific wavelength of light,
using confocal microscope.
Image Acquisition

15.  Green spots = Genes
upreguated compared
to control
 Red Spots = Genes
downregulated in
experimental sample
 Yellow Spots = Genes of
equal abundance in
both experimental &
control sample
Data Analysis
Source:http://libertgen564s15.weebly.com/microarray.html
Source:https://deutsch.physics.ucsc.edu/micr
oarrays.html

17. To answer some of fundamental questions in
biology such as “when, where and to what
magnitude genes of interest are expressed.”
Measuring transcript abundance (cDNA arrays)
Genotyping
Estimating DNA copy number (CGH)
Wide spread use in gene mutation analysis to
analyze genomic alteration.
Application