DNA MICROARRAY

Submitted by: Romila

Submitted to: 7857
Dr. Pooja Arora Group-1
Department of Zoology B.Sc. Hons. Zoology
Hansraj College Semester 6
Hansraj College
University of Delhi
CONTENTS
• Introduction
• Principle
• Why do we do it?
• Basic Procedure
• Types
• cDNA based microarrays
• Oligonucleotide based microarrays
• Data Analysis
• Limitations of microarrays
• Applications
• Research Papaer
• References
A DNA microarray (DNA chip, or gene chip) experiment consists of hybridizing a
nucleic acid sample (target) derived from the messenger RNAs (mRNAs) of a
cell or tissue to single-stranded DNA sequences (probes) that are bound in an
ordered arrangement to a solid platform.
 PRINCIPLE
• Core principle behind microarrays is
hybridization between two DNA strands, the
property of complementary nucleic acid
sequences to specifically pair with each other
by forming hydrogen bonds between
complementary nucleotide base pairs.
• After washing off non-specific bonding
sequences, only strongly paired strands will
remain hybridized. 
• Fluorescently labeled target sequences that
bind to a probe sequence generate a signal. 
• Total strength of the signal, from a spot
(feature), depends upon the amount of target
sample binding to the probes present on that
spot.
 Why do we do it ?
Measures the expression levels of not a few but
rather thousands of genes in the same experiment.

Thousands of single-stranded sequences that are

complementary to target sequences are bound,
synthesized, or spotted to a glass support whose
size is similar to a typical microscope slide.

Designed to measure the transcriptional levels of

RNA transcripts derived from thousands of genes
within a genome in a single experiment.

These DNA sequences are compared with each

other to get the needed information.
Basic Procedure

1.Collect samples
2.Isolate mRNA
3.Create labelled DNA
4.Hybridization
5.Microarray Scanner
6.Analyze Data
 TYPES

1) cDNA based microarrays

2) Oligonucleotide based microarrays

The design of the probes (probe set) for a microarray depends on the objective of the
experiment and the degree of resolution that is required.

Computer programs determine probe sequences that are specific for their target
sequences, are least likely to hybridize with nontarget sequences (cross-hybridize), have
no secondary structure (foldback) that would prevent hybridization with the target
sequence, and have similar melting (annealing) temperatures, so that all target
sequences can bind to their complementary probe sequences under the same conditions.
 1) cDNA based microarrays
DNA microarray is constructed by spotting polymerase chain reaction (PCR)-amplified cDNA sequences
from the mRNAs of a single cell or all or specific sets of the coding sequences of an organism onto a glass
slide or nylon membrane. In this case, about 10,000 different probes can be arrayed in a 1-cm2 area.
Procedure
• mRNA is extracted from two samples (sample 1 and sample 2), and reverse transcripted to form cDNA.
• The first cDNA strands are
labeled with the fluorescent dyes
Cy3 and Cy5, respectively.
• The cDNA samples are mixed and
hybridized to an ordered array of
either gene sequences or gene-
specific oligonucleotides.
• After the hybridization reaction,
each probe cell is scanned for
both fluorescent dyes and the
separate emissions are recorded.
• Probe cells that produce only a
green or red emission represent
genes that are transcribed only in
samples 1 and 2, respectively;
yellow emissions denote genes
that are active in both samples;
and no emissions (black) rep-
resent genes that are not
transcribed in either sample.
Limitations of cDNA based microarrays

1.Limitation of using large cDNA sequences is an uneven optimal

melting temperature caused by differences in their sizes and the content
of GC-paired nucleotides. 
2. Cross-hybridization of closely related sequences, overlapped genes,
and splicing variants takes place.

The oligonucleotides are designed in such a way to avoid the cDNA probe
drawbacks and to maximize the specificity for the target gene.
 2) Oligonucleotide microarrays
• Utilizes sets of oligonucleotides as probes, usually
representing thousands of genes.
• For one commonly used platform, the probes are
synthesized directly (in situ) on a solid surface (quartz
wafer) by a light-directed process known as
photolithography using blockers and masks.
• Thousands of copies of an oligonucleotide with the
same specific nucleotide sequence are synthesized in
a predefined position (probe cell or feature) on the
array surface.
• The probes are typically 10 to 40 nucleotides, and
several probes with different sequences for each gene
will be synthesized on the microarray.
• Longer oligonucleotides up to 100 nucleotides can also
be used.
• A complete whole-genome oligonucleotide array may
contain more than 500,000 probes representing as
many as 30,000 genes.
Procedure

• mRNA is purified with a poly(dT) sequence that has a T7 RNA polymerase primer
sequence extension. 
• After two-stranded cDNA synthesis, the second cDNA strand acts as a template
for synthesis of cRNA by T7 RNA polymerase in the presence of ATP, cytidine
triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP),
biotinylated CTP, and biotinylated UTP. 
• The gray circles represent incorporated biotinylated nucleotides. 
• The biotinylated cRNA is purified, fragmented into pieces from 50 to 100
nucleotides in length, and hybridized to an oligonucleotide microarray. 
• The microarray is treated with streptavidin–phycoerythrin, and the probe cells
(black squares) are scanned for emission (yellow) from the biotin-bound
streptavidin–phycoerythrin.
 Analysis of Data
•The response to a biological condition is determined by
comparing the fluorescence intensity for each gene (each
probe cell), averaged among replicates, under two
different conditions and calculating the ratio, commonly
expressed as an n-fold change.
•The log ratios for all probe cells are compiled into a table
called an expression matrix.
•For a clear presentation of the clustered data, ranges of
log ratio values are assigned arbitrary colors. Usually,
black is designated for a log ratio of zero, dark to bright
red for increasing positive log ratios, and dark to bright
green for decreasing negative log ratios. 
•In other words, red is frequently used to denote gene
overexpression and green to denote underexpression.
•A visualized representation of a clustered microarray is
called a gene expression profile, where the rows
represent the reordered genes and the columns represent
either conditions or samples.
Limitations of DNA microarrays
• The results take a lot of time to analyze as the amount of data collected from
each array will be huge
• The results may be too complex to interpret and are not always quantitative
• The results are not always reproducible
• The technology is too expensive
• The arrays provide an indirect measure of relative concentration
• Especially for complex mammalian genomes, it is often difficult to design
arrays in which multiple related DNA/RNA sequences do not bind to the same
probe on the array
• A DNA array can only detect sequences that the array was designed to
detect.
• Misleading interpretations are caused.
• Experiment can not be carried out without any prior knowledge of the RNAs
that are present, unable to provide information on a transcriptome that has not
been previously characterized.
Applications

•Drug discovery
• Study of functional genomics
• DNA sequencing
• Gene expression profiling
• Study of proteomics
• Diagnostics and genetic engineering
• Toxicological researches
• Pharmacogenomics and theranostics.
HOW EPSTEIN-BARR VIRUS SETS OFF MULTIPLE SCLEROSIS
 Multiple Sclerosis
A disease in which the immune system eats away at the protective covering of nerves.
In MS, resulting nerve damage disrupts communication between the brain and the body

New research shows that the Epstein-Barr virus, a common type of herpes virus,
triggers multiple sclerosis by priming the immune system to attack the body’s own
nervous system.
Myelin forms the protective coating around nerve cells, and when it’s damaged, electrical
impulses can no longer jump efficiently from one nerve to the next, resulting in the
numbness, muscle weakness, and severe fatigue of multiple sclerosis.

Previous research had shown that multiple sclerosis patients have increased antibodies to a variety
of common viruses, including measles, mumps, varicella-zoster, and Epstein-Barr virus. But
despite this epidemiologic correlation, scientists struggled to prove a clear connection.
• The researchers started by examining the antibodies produced by immune cells in the blood
and spinal fluid of nine MS patients.
• Unlike in healthy individuals, the immune cells of MS patients traffic to the brain and spinal
cord, where they produce large amounts of a few types of antibodies. Patterns of these
antibody proteins, called oligoclonal bands, are found during analysis of the spinal fluid and
are part of the diagnostic criteria for MS.
• They analyzed the antibodies from the oligoclonal bands, and showed that they come from B c
the spinal fluid.
• They expressed individual antibodies from these cells and tested them for reactivity against
hundreds of different antigens.
• They started with human antigens, but couldn’t find clear reactivity. So eventually they tested t
against EPSTEIN-BARR VIRUS (EBV) and other herpes viruses,several of these antibodies, a
one in particular, bound to EBV.
• Six of the nine MS patients had antibodies that bound to the EBV protein EBNA1, and eight of
had antibodies to some fragment of EBNA1.
• Next, the researchers tested the same antibody
on a microarray containing more than 16,000
human proteins.
• Part of the EBV protein mimics our own host
protein—in this case, GlialCAM, found in the
insulating sheath on nerves.
• When they discovered that the antibody also
bound with high affinity to GlialCAM, they
knew they’d found a specific mechanism for
how EBV infection could trigger multiple
sclerosis.

• When the immune system attacks EBV to

clear the virus, it also ends up targeting
GlialCAM in the myelin
 CONSEQUENCES

• The most exciting aspect of this discovery is its potential to create new pathways for
the clinical treatment of multiple sclerosis.
• A vaccine against Epstein-Barr virus could perhaps eventually eradicate MS, in the
same way polio was eradicated.
• This research also demonstrates why manufacturers would need to be extra careful in
selecting which antigens to incorporate into an EBV vaccine. Antigens, like EBNA1
have tobe avoided, that could cause autoimmunity.
• The discovery of how EBV triggers multiple sclerosis could also have ramifications
for research into other autoimmune diseases, such as lupus and rheumatoid arthritis,
which, like MS, have been significantly associated with EBV infection in
epidemiologic studies.
Q1) Which of the following statements is incorrect?
a) Usually black colour is used to designate log ratio of zero.
b) Red is frequently used to denote gene overexpression.
c) Green is used for increasing positive log ratios
d) Decreasing negative log ratios denote underexpresssion of gene.
Q2) While using large target sequences to study the genome of an organism,
which method would be best employed?
a) cDNA based microarrays
b) Oligonucleotide based microarrays
c) Western or Southern blotting techniques
Q3) Why is mRNA purified with a poly (dT) sequence?
a)To aid biotinylation of cRNA
b) To aid the reverse transcription process
c) Streptavidin binds to the deoxythymine and elicits emissions
during laser scanning
d) None of the above
Q4) For which purpose DNA microarray can not be used?
a) To study the up and down regulation of genes due to a disease
b) Comparison of genes of yeast grown under aerobic and anaerobic
conditions
c) To determine which genes are expressed at certain stages of development
d) Provide information on a transcriptome that has not been previously
characterized
Q5) How does EBV cause Multiple Sclerosis?
a) EBV eats away the GlialCAM protein in the myelin sheath of the
neurons.
b) EBV protein mimics our own host protein, GlialCAM, found in the
insulating sheath on nerves.
c) EBV blocks neural transmission signals between brain and body.
d) The protein EBNA1 in EBV is toxic for neural cells.
Answers
Q1) c
Q2) b
Q3) b
Q4) d
Q5) b
References

• Molecular Biotechnology: Principles and Applications of Recombinant DNA.

Book by Bernard R. Glick and Jack J. Pasternak,fourth edition
• GENE CLONING AND DNA ANALYSIS: An Introduction by T.A. BROWN
University of ManchesterManchester, Seventh Edition
• https://molmed.biomedcentral.com/articles/10.2119/2006-00107.Trevino
• https://www.news-medical.net/amp/life-sciences/DNA-microarray.aspx
• https://www.nature.com/articles/s41586-022-04432-7
THANK YOU