Miarray:A new Technology For Life Science

2. Course code:MPG-521
Seminar presentation
Presented by:
Md.Abdul Momin
201-010-061
Department of Microbiology
Primeasia University
Miroarry: A new Technology For Life Science

3. 1. INTRODUCTION
2. HISTORY
3. PRINCIPLE
4. DNA MICROARRAY TECHNOLOGY
5. PRINCIPLES OF DNA MICROARRAY TECHNOLOGY
6. TYPES OF DNA MICROARRAY
 GLASS cDNAMICROARRAYS
 IN SITU OLIGONUCLEOTIDEARRAY FORMAT
7.APPLICATIONS OF MICROARRAY TECHNOLOGY
CONTENTS:

4. INTRODUCTION:
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
RNAin a sample.
DNA microarray uses a million different probes, fixed on
a solid surface.

5. 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.
What is An Array
Fig-Robotic arm with spotting slides

6. HISTORY
 Microarray technology evolved from Southern blotting.
 The concept of microarrays was first proposed in the late
1980s byAugenlicht and his colleagues.
 They spotted 4000 cDNAsequences 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

7. PRINCIPLE
 The core principle behind
microarrays is hybridization
between two DNAstrands.
 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- Array hybridization

8. DNA MICROARRAY TECHNOLOGY
 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.
 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. PRINCIPLES OF DNA MICROARRAY
TECHNOLOGY:
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.

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 reversetranscriptase
enzyme.
 Short primer is required to initiate
cDNAsynthesis.
Each cDNA(Sample and Control) is
labelled with fluorescent cyanine
dyes (i.e. Cy3 and Cy5).
SAMPLE RREPARATION AND LABELING
Fig- 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.
ARRAY HYBRIDISATION
Fig-Array Hybridisatio

13. TYPES OF DNA MICROARRAY
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.
 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).
GLASS cDNA MICROARRAYS
Fig-Contact printer with robotic pins

14. 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.
Advantages of Glass cDNA microarrays:
Glass cDNA microarray have a few disadvantages such as
intensive labour requirement for synthesizing, purifying, and
storing DNAsolutions before microarray fabrication.
They may hybridise to spots designed to detect transcript
from a different gene.
Disadvantages of Glass cDNA microarrays

15.  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.
IN SITU OLIGONUCLEOTIDE
ARRAY FORMAT:

16.  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.
In Situ Light-Directed Oligonucletide
Probe Array Synthesis:
Fig-Photolithography process

17. Advantages offered by the in situ oligonucleotide array
format include speed, specificity and reproducibility.
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.
Advantages In of situ oligonucleotide
array formats
Diadvantages In of situ oligonucleotide array
formats

19.  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 .
Microarray as a Gene Expression
Profiling Tools:

20.  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.
Microarray as a Comparative
Genomics Tools:

21.  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.
Disease Diagnosis:
 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.
Drug Diacovery:

22. 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.
Toxicological Research:

23. 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.
Conclusion: