TOPIC : MICROARRAYS

Presented by ;
somama Khabeer
Ubaid Razzaq

Course instructor ;
Sir Nasir Khan
 Content
• Introduction
• Types of Microarrays
• Cellular Microarrays
• Peptide Microarrays
• DNA Microarrays
• Application of Microarrays
 MICROARRAY TECHNOLOGY

•A microarray is a laboratory toolusedto detect the

expression of thousands of genes at the same time. DNA
microarrays are microscope slides that are printed with
thousands of tiny spots in defined positions, with each
spot containing a known DNA sequence or gene . A
laboratory tool used to analyze large numbers of genes
or proteins at one time. In a microarray , biologic
molecules such as DNA, RNA, or protein are placed in a
pattern onto a surface such as a glass slide.
 Chemical compound microarray

• A chemical compound microarray is a collection of

organic chemical compounds spotted on a solid
surface, such as glass and plastic. This microarray
format is very similar to DNA microarray, protein
microarray and antibody microarray. In chemical
genetics research.
• they are routinely used for searching proteins that
bind with specific
• chemical compounds, and in general drug
discovery research
• Form of chemical compound microarray
• There are three different forms of chemical
compound microarrays based on the
fabrication method.1. The first form is to
covalently immobilize the organic compounds
on the solid surface with diverse linking
techniques this platform is usually called
Small Molecule Microarray
 Form of Chemical compound microarray

• The second form is to spot and dry organic

compounds on the solid surface without
immobilization, this platform has a commercial
name as Micro Arrayed Compound Screening
• Last form is to spot organic compounds in a
homogenous solution without immobilization
and drying effect.
 Cellular microarray

•A cellular microarray (cell microarray) is a

laboratory tool that allows for the multiplex
interrogation of living cells on the surface of a
solid support.
•The support, sometimes called a"chip",is
spotted with varying materials, such as
antibodies, proteins, or lipids, which can
interact with the cells, leading to their capture
on specific spots.
 Type of cellular microarray

1.Reverse transfection
• •Reverse transfection is a technique for the
transfer of genetic material into cells.
2. PMHC Cellular Microarrays.
• •PMHC cellular microarrays are a type of cellular
microarray that has been spotted with pMHC
complexes peptide-MHC class 1or peptide-MHC
class 2These biochips can be used to interrogate
immune cells
 Peptide microarray

•A peptide microarray also commonly known as peptide

chip or peptide epitope microarray is a collection of
peptides displayed on a solid surface, usually a glass or
plastic chip.

•Peptide chips are used by scientists in biology,

medicine and pharmacology to study binding
properties and functionality and kinetics of protein-
protein interactions in general.
 Peptidemicroarray

•In basic research, peptide microarrays are often

used to profile an enzyme (like kinase,
phosphatase, protease, acetyltransferase,
histone deacetylase etc.), to map an antibody
epitope or to find key residues for protein
binding.
 Principle

•The assay principle of peptide microarrays is similar to an

ELISA protocol. The peptides are linked to the surface of a
glass chip
• typically the size and shape of a microscope slide. This
peptide chip
• can directly be incubated with a variety of different biological
• samples like purified enzymes or antibodies, patient or animal
sera,
• cell lysates and then be detected through a label-dependent
fashion.
 AdvantagesPeptide
• microarrays show several advantages over protein
microarrays
•Ease and cost of synthesis

•Extended shelf stability

•Flexible design for peptide sequence

•Higher batch-to-batch reproducibility

 Applications of peptide microarrays

•Peptide microarrays can be used to study different kinds

of protein-

• protein interactions specially those involving modular

protein substructures called peptide recognition
modules or, most commonly, protein interaction
domains.
•Identification of substrates for orphan enzymes
•Optimization of known enzyme substrates
•Detection of contaminating enzyme activities
 DNA microarrays
 A DNA microarray is a collection of microscopic
DNA spots attached to a solid surface. Each DNA
spot contains many thousands of copies of a
specific DNA sequence, known as probes. These
usually correspond to a short section of a gene –
generally at the 3′ end. Each microarray includes
one or a few probe sets for each interrogated
gene. These are used to hybridize a cDNA sample
(the target) under high-stringency conditions.
•  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.
• The original nucleic acid arrays were macro arrays
approximately 9 cm × 12 cm and the first
computerized image based analysis was published in
1981
• .An example of its application is in SNPs arrays for
polymorphisms in cardiovascular diseases, cancer,
pathogens and GWAS analysis. Also for identification
of structural variations and measurement of gene
expression.
 Principle
• The principle of DNA microarray technology is
based on fact that complementary sequences
of DNA can be used to hybridise immobilised
DNA molecule
• There are four major step
I. Simple preparation and labeling
II. Hybridization
III. Washing
IV. Image acquisition and data analysis
 preparation and labeling
• Isolate a total RNA containing mRNA that
ideally represent a quantitative copy of genes
expressed at time of simple collection
• Preparation of cDNA from mRNA using a
reverse transcriptase enzyme
• Primer is required to initiate cDNA synthesis
• Each cDNa is labeled with florescent cyanine
dyes
Preparation and labeling
 Array hybridization
• Here the labeled cDNA (sample and control)
are mixed together
• Purification
• After purification the mixed labeled cDNA is
competitively hybridized against denatured
PCR product or cDNA molecule spotted on a
glass
Array hybridization
Image acquisition and data analysis
• Slide is derived and scanned to determine how
much labeled cDNA probes is bound to each
target spot
• Hybridization target produces emission
• Microarray software often use green spot on the
microarray to represent unregulated genes
• Red to represent those genes that are down
regulated and yellow to present in equal
abundance
 Types of DNA microarrays
• Glass cDNA
• High density oligonucleotide microarrays
 Types of Microarrays
• Depending upon the kind of immobilized
sample used construct arrays and the
information fetched, the Microarray
experiments can be categorized in three ways:
  Microarray Expression Analysis
 Microarray for Mutation Analysis
 Comparative Genomic Hybridization
  Microarray Expression Analysis
• In this experimental setup, the cDNA derived
from the mRNA of known genes is immobilized.
• The sample has genes from both the normal as
well as the diseased tissues. Spots with more
intensity are obtained for diseased tissue gene
if the gene is over expressed in the diseased
condition.
• This expression pattern is then compared to the
expression pattern of a gene responsible for a
disease
 Microarray for Mutation Analysis
•  For this analysis, the researchers use gDNA.
The genes might differ from each other by as
less as a single nucleotide base.
• A single base difference between two
sequences is known as Single Nucleotide
Polymorphism (SNP) and detecting them is
known as SNP detection.
  Comparative Genomic Hybridization:

•  It is used for the identification in the increase

or decrease of the important chromosomal
fragments harboring genes involved in a
disease.
 Applications of Microarrays
• Gene Discovery: DNA Microarray technology
helps in the identification of new genes, know
about their functioning and expression levels
under different conditions.
• Disease Diagnosis
• Drug Discovery
• Toxicological Research
• In cancer
• Antibiotic treatment
• Early detection of oral precancerous lesions
 Refrances
• https://academic.oup.com/clinchem/article/4
7/8/1479/5639454
• https://en.wikipedia.org/wiki/DNA_microarra
y
• https://www.sciencedirect.com/sdfe/pdf/dow
nload/eid/3-s2.0-B9780123859440000022/firs
t-page-pdf