CSU1110 – Genomics, Proteomics and Metabolomics

Lecture 17 – DNA Microarrays

 Topics to be covered today
1.Introduction to array
2. Introduction to DNA microarray
– Definition
– Types
– Steps involved in microarray
– Applications
3. Conclusion
 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 RNA in a sample.

 DNA microarray uses a million different probes, fixed on a solid surface.

 WHAT IS AN ARRAY

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
 HISTORY

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.
 PRINCIPLE

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

 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.
 STEPS 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.

Sample preparation Image acquisition

and Hybridisation Washing and
labeling Data analysis
 SAMPLE PREPARATION AND LABELING

 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 Fig-03 Sample labeling

(i.e. Cy3 and Cy5).

 ARRAY HYBRIDISATION

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.
 IMAGE ACQUISITION AND DATA ANALYSIS

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
 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

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
 MANUFACTURING OF GLASS cDNA
MICROARRAY

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.
FIG-07 Spotting of
slides
ADVANTAGES OF GLASS cDNA MICROARRAYS

 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 specialized equipment.

 Data capture can be carried out using equipment that is very often already
available in the laboratory.
 DISADVANTAGES OF GLASS cDNA MICROARRAYS

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.
 IN SITU OLIGONUCLEOTIDE ARRAY FORMAT

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 light-directed oligonucleotide probe array synthesis.

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
Fig-08 Photolithography
containing a photolabile protecting process

group X. This process is repeated until

the desired product is obtained.
 ADVANTAGES OF IN
SITU OLIGONUCLEOTIDE ARRAY FORMAT

• Advantages offered by the in situ oligonucleotide array format include speed,

specificity and reproducibility.
 DISADVANTAGES OF
IN SITU OLIGONUCLEOTIDE ARRAY FORMAT

In situ oligonucleotide array formats tend to have expensive specialized

equipments e.g. to carry out the hybridization, staining of label, washing, and
quantitation process.

Short-sequences used on the array have decreased sensitivity/binding compared

with glass cDNA microarrays.
 Applications of Microarray Technology

MICROARRAY MICROARRAY
AS A GENE AS A DISEASE DRUG TOXICOLOGICA
EXPRESSION COMPARATIV L RESEARCH
PROFILING E GENOMICS DIAGNOSIS DISCOVERY
TOOL TOOL
 MICROARRAY AS A GENE EXPRESSION
PROFILING TOOL
• 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 COMPARATIVE
GENOMICS TOOL
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.
 DISEASE DIAGNOSIS

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.
 DRUG DISCOVERY

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.
 TOXICOLOGICAL RESEARCH

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.
Thank you

Dr. Rupak Nagraik

School of Bioengineering and Food Technology
Shoolini University
Village Bhajol, Solan (H.P)

7018858252 (Mob No.)

rupaknagraik@shooliniuniversity.com