1. DNA MICROARRAYS
ARUNIMA A G
I M TECH BIOTECHNOLOGY

2. 1. INTRODUCTION
2. HISTORY
3. PRINCIPLE
4. DNA MICROARRAY TECHNOLOGY
5. PRINCIPLES OF DNA MICROARRAY
TECHNOLOGY
6. TYPES OF DNA MICROARRAY
 GLASS cDNA MICROARRAYS
 IN SITU OLIGONUCLEOTIDE ARRAY
FORMAT
7. APPLICATIONS OF MICROARRAY
TECHNOLOGY

3. The large-scale genome sequencing effort and the
ability to immobilize thousands of DNA fragments on
coated glass slide or membrane, have led to the
development of microarray technology.
A microarray is a pattern of ssDNA probes which are
immobilized on a surface called a chip or a slide.
 Microarrays use hybridization to detect a specific DNA
or RNA in a sample.
 DNA microarray uses a million different probes, fixed
on a solid surface.

4.  An array is an orderly
arrangement of samples where
matching of known and
unknown DNA samples is done
based on base pairing rules.
 An array experiment makes use
of common assay systems such
as microplates or standard
blotting membranes.
Fig-01 Robotic arm with
spotting slides

5.  Microarray technology evolved from Southern
blotting.
 The concept of microarrays was first proposed in the
late 1980s by Augenlicht and his colleagues.
 They spotted 4000 cDNA sequences on nitrocellulose
membrane and used radioactive labeling to analyze
differences in gene expression patterns among
different types of colon tumors in various stages of
malignancy.

6.  The core principle behind
microarrays is hybridization
between two DNA strands.
 Fluorescent labeled target
sequences that bind to a probe
sequence generate a signal that
depends on the strength of the
hybridization determined by
the number of paired bases.
Fig-02 Array hybridization

7.  DNA microarray technology may be defined as a
high-throughput and versatile technology used for
parallel gene expression analysis for thousands of
genes of known and unknown functions.
 Used for detection of polymorphisms and mutations
in genomic DNA
 A DNA microarray is a collection of microscopic
DNA spots on solid surface. Each spot contains
picomoles of a specific DNA sequence, known as
probes or reporters.

8.  Each identified sequenced gene on the glass, silicon
chips or nylon membrane corresponds to a fragment
of genomic DNA, cDNAs, PCR products or
chemically synthesized oligonucleotides of up to
70mers and represents a single gene.
 Probe-target hybridization is usually detected and
quantified by detection of fluorophore, silver, or
chemiluminescence labeled targets to determine
relative abundance of nucleic acid sequences in the
target.

10.  The principle of DNA microarray technology is based
on the fact that complementary sequences of DNA
can be used to hybridise, immobilised DNA
molecules.
 There are four major steps in performing a typical
microarray experiment.
Sample preparation
and
labeling
Hybridisation Washing
Image acquisition
and
Data analysis

11.  Isolate a total RNA containing
mRNA that ideally represents a
quantitative copy of genes
expressed at the time of sample
collection.
 Preparation of cDNA from
mRNA using a reverse-
transcriptase enzyme.
 Short primer is required to initiate
cDNA synthesis.
 Each cDNA (Sample and Control)
is labelled with fluorescent
cyanine dyes (i.e. Cy3 and Cy5). Fig-03 Sample labeling

12.  Here, the labelled cDNA
(Sample and Control) are
mixed together.
 Purification
 After purification, the
mixed labelled cDNA is
competitively hybridised
against denatured PCR
product or cDNA molecules
spotted on a glass slide.
Fig-04 Array Hybridisation

13.  Slide is dried and scanned to
determine how much labelled
cDNA (probe) is bound to
each target spot.
 Hybridized target produces
emissions.
 Microarray software often uses
green spots on the microarray
to represent upregulated genes.
 Red to represent those genes
that are downregulated and
yellow to present in equal
abundance
Fig-05 Gene chip showing different
type of color spots

14. 1) Glass cDNA microarrays which involves the micro
spotting of pre-fabricated cDNA fragments on a glass
slide.
2) High-density oligonucleotide microarrays often
referred to as a "chip" which involves in
situ oligonucleotide synthesis.

15.  Glass cDNA microarrays
was the first type of DNA
microarray technology
developed.
 It was pioneered by Patrick
Brown and his colleagues at
Stanford University.
 Produced by using a robotic
device which deposits
(spots) a nanoliter of DNA
onto a coated microscopic
glass slide (50-150 µm in
diameter) .
Fig-06 Contact printer with
robotic pins

16. FIG-07 Spotting of
slides
Selection of the material to
spot onto the microscope glass
surface.
Preparation and purification of
DNA sequences representing
the gene of interest.
Spotting DNA solution onto
chemically modified glass
slides via a contact printing or
inkjet printing.

17.  Advantages of Glass cDNA microarrays include their
relative affordability with a lower cost.
 Its accessibility requiring no specific equipment for use
such that hybridisation does not need specialised
equipment.
 Data capture can be carried out using equipment that is
very often already available in the laboratory.

18.  Glass cDNA microarray have a few disadvantages such
as intensive labour requirement for synthesizing,
purifying, and storing DNA solutions before microarray
fabrication.
 They may hybridise to spots designed to detect
transcript from a different gene.

19.  Oligonucleotides are synthesized on the chip.
 Presently, the commercial versions of Affymetrix Gene
Chips hold up to 500,000 probes/sites in a 1.28-cm2
chip area.
 Due to such very high information content (genes) they
are finding widespread use in the hybridisation-based
detection and analysis of mutations and
polymorphisms, such as single nucleotide
polymorphisms.

20.  Light is directed through a
photolithographic mask to
specific areas of array surface.
 Activation of areas for chemical
coupling. Attachment of A
nucleotide containing photolabile
protecting group X (MeNPOC).
 Next light is Directed to a
different region of the array
surface through a new mask.
 Addition of 2nd building block T
containing a photolabile
protecting group X. This process
is repeated until the desired
product is obtained.
Fig-08 Photolithography
process

21.  Advantages offered by the in
situ oligonucleotide array format include
speed, specificity and reproducibility.

22.  In situ oligonucleotide array formats tend to have
expensive specialised equipments e.g. to carry out the
hybridisation, staining of label, washing, and
quantitation process.
 Short-sequences used on the array have decreased
sensitivity/binding compared with glass cDNA
microarrays.

23. MICROARRA
YAS A GENE
EXPRESSION
PROFILING
TOOL
MICROARRA
YAS A
COMPARATIV
E GENOMICS
TOOL
DISEASE
DIAGNOSIS
DRUG
DISCOVERY
TOXICOLOGIC
AL RESEARCH

24.  The principle aim of using microarray technology as a gene
expression profiling tool is to answer some of the fundamental
questions in biology such as "when, where, and to what
magnitude genes of interest are expressed.
 Microarray analysis measure changes in the multigene patterns
of expression to better understand about regulatory
mechanisms and broader bioactivity functions of genes.

25.  Microarray technology have widespread use in
comparative gene mutation analysis to analyse
genomic alterations such as sequence and single
nucleotide polymorphisms.
 In microbiology microarray gene mutation analysis is
directed to characterisation of genetic differences
among microbial isolates, particularly closely related
species.

26.  Different types of cancer have been classified on the
basis of the organs in which the tumors develop.
 Now, with the evolution of microarray technology, it
will be possible for the researchers to further classify
the types of cancer on the basis of the patterns of gene
activity in the tumor cells.

27.  Microarray technology has extensive application in
Pharmacogenomics.
 Comparative analysis of the genes from a diseased and
a normal cell will help the identification of the
biochemical constitution of the proteins synthesized by
the diseased genes.

28.  Microarray technology provides a robust platform for the
research of the impact of toxins on the cells and their passing
on to the progeny.
 Toxicogenomics establishes correlation between responses to
toxicants and the changes in the genetic profiles of the cells
exposed to such toxicants.
 The microarray permits researchers to examine thousands of
different genes in the same experiment and thus to obtain a
good understanding of the relative levels of expression
between different genes in an organism.

29.  Microarray is a recently developed functional genomics
technology that has powerful applications in a wide
array of biological medical sciences, agriculture,
biotechnology and environmental studies. Since many
universities research institutions and industries have
established microarray based core facilities and
services, microarrays have become a readily accessible,
widely used technology for investigating biological
systems.

30. THANK YOU