VIKAS KUMAR SINGH

CHAUDHARY CHARAN SINGH UNIVERSITY,

MEERUT

ASSIGNMENT ON
MICROARRAY

SUBJECT
PLANT BIOTECHNOLOGY

SUBMITTED TO
DR. SHAILENDRA SHARMA SIR
DEPARTMENT OF GENETICS & PLANT BREEDING,
CHAUDHRY CHARAN SINGH UNIVERSITY,
MEERUT

SUBMITTED BY

VIKAS KUMAR SINGH

M.SC. (AG) IIIrd SEM
DEPARTMENT OF GENETICS AND PLANT BREEDING
CHAUDHARY CHARAN SINGH UNIVERSITY MEERUT
2018
 VIKAS KUMAR SINGH

MICROARRAY
Microarray” has become a general term; there are many types now,

 DNA microarrays.
 Protein microarrays.
 Transfection microarrays.
 Antibody microarray.
 Tissue microarray.
 Chemical compound microarray.
We’ll be discussing DNA microarrays

DNA microarray

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. DNA
Microarray is a technique for genome Analysis, where thousands of genes are analyzed at a time
in a single experiment simultaneously to determine which genes are expressed in a particular
sample.

 These experiments rely on the principle of hybridization.

 Thousands of known probes are fabricated onto a small solid slide (Glass, Silicon,
and Nylon) in a particular order as spots and called DNA microarray or DNA chip.
 The spots can be DNA, cDNA, or oligonucleotides.
 cDNA microarray was first introduced by Patrick Browns lab in 1995.
 Oligo microarray was introduced by Affimatrix in 1996.

Mark Schena Patrick O Brown

Genetic Content of Microarrays:-

An immobilized nucleic acid sequence on the microarray is
place where the genetic content of microarrays occupies. And by its identity e can determine the
information from it and also clarify that, how reliable this information is. As microarrays enable
simultaneous interrogation of up to tens or hundreds of thousands of targets with one or more
labeled probes, generation of accurate data demands that only specific interactions result in
detectable signals. Several strategies for preparing the immobilized target nucleic acids for
microarrays exist.
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These nucleic acids can be synthesized directly on the microarray or they can be purified cDNA
clones, other DNA fragments or oligonucleotides, which are deposited onto the array by a printing
process. This flexibility of using either partially characterized sequences or defined
oligonucleotides as targets has improved the application of microarray analysis to different
biological problems in a number of species.

Sources of microarray target,

Types of Microarray
There are major two types of Microarray:
 cDNA Microarray.
 Oligonucleotide.

1. cDNA Microarray:-
 cDNA Microarray, first introduced by Patrick Brown, is also termed as spotted
microarray.
 In this technique, DNA fragments (genes) are amplified by PCR and then spotted
onto a glass slide in a grid like pattern.
 This is a two-channeled microarray that is typically hybridized with cDNA from
two samples to be compared on the same slide for visualization of genes.
 These samples are fluorescently labeled (Cys 3-rhodamine for red and Cys 5-
fluorescin for green) for easy detection.
 Data from a cDNA microarray provides information about the relative expression
of genes in two different biological samples.
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What is the cDNA?

To study specific events in development, cell death, cancer, and other
biological processes, a library of the subset of the genome that is expressed in a given cell type at
a given time can be a valuable tool. Genomic libraries and chromosome libraries contain all the
genes in a genome or on a chromosome, but these collections cannot be directly used to find genes
that are actively expressed in a cell. A cDNA library contains DNA copies made from the
messenger RNA (mRNA) molecules present in a cell population at a given time, and therefore
represents the genes being expressed in the cells at the time the library was made. It is called a
cDNA library because the DNA it contains known as complementary DNA, or cDNA is
complementary to the nucleotide sequence of the mRNA.
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 Firstly, the mRNA is obtained and purified from the rest of the RNAs. Several methods exist for
purifying RNA such as trizolextraction and column purification. Column purification is done by
using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will
bind. The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer and some
heat to separate the mRNA strands from oligo-dT.
 Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymidine nucleotides) is tagged as
a complementary primer which binds to the poly-A tail providing a free 3'-OH end that can be
extended by reverse transcriptase to create the complementary DNA strand. Now, the mRNA is
removed by using a RNAse enzyme leaving a single stranded cDNA (sscDNA). This sscDNA is
converted into a double stranded DNA with the help of DNA polymerase. However, for DNA
polymerase to synthesize a complementary strand a free 3'-OH end is needed. This is provided by
the sscDNA itself by generating a hairpin loop at the 3' end by coiling on it. The polymerase
extends the 3'-OH end and later the loop at 3' end is opened by the scissoring action
of S1 nuclease. Restriction endonucleases and DNA ligase are then used to clone the sequences
into bacterial plasmids. The cloned bacteria are then selected, commonly through the use of
antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown
and sequenced to compile the cDNA library.

2. Oligonucleotide Microarray:-
 In this method, a DNA microarray slide is fabricated by synthesizing 11-16 short
oligonucleotides (approx. 25bp in length) at very high density for each expressed
gene on a slide.
 Here oligonucleotides are synthesised at fixed locations by photolithographic
technique and chemical synthesis technique.
 In Affimatrix, a 25 bp long oligos are taken and a total of 100 light masks are
made for each base at each layer.
 An oligos-MA contains two sets of probes for each gene one set for perfect
match and another that contains mismatch nucleotides.
 Here two or more samples can be compared at a time but each sample needs
different microarray slides.
 These arrays are used in genotyping experiments, where sequences of alternate
gene forms are synthesised to detect polymorphisms and mutations.
 A white spot is generated for strong hybridization and no hybridization produces
black spots.
 In between these two colours is marginal hybridization.
 Oligonucleotide microarrays avoid the cross- hybridization that may occur with
the cDNA microarray.
 They are much expensive and also need very accurate gene annotation to
fabricate match and mismatch probes.
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Comparison: cDNA vs Oligonucleotide Microarrays,

Microarray Fabrication Technologies

1. Photolithography (Affymetrix, Oligonucleotide Microarray).

2. Contact printing (Patrick O Brown, Stanford University).
3. Non-Contact Printing (Pin and Ring, Bubble Jet, Ink Jet).

1. Photolithography (Affymetrix, Oligonucleotide Microarray).

Photolithography: UV light
is passed through a lithographic mask that acts as a filter to either transmit or block the light from
the chemically protected microarray surface (wafer). The sequential application of specific
lithographic masks determines the order of sequence synthesis on the wafer surface. (Bottom)
Chemical synthesis cycle. UV light removes the protecting groups (squares) from the array
surface, allowing the addition of a single protected nucleotide as it is washed over the microarray.
Sequential rounds of light deprotection, changes in the filtering patterns of the masks, and single
nucleotide additions form microarray features with specific 25-bp probes.
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Roche Nimblegen Oligonucleotide Microarray photolithography: -

He utilizes a digital
micromirror device (DMD) to create virtual masks. The DMD forms the pattern of UV light
needed to direct the specific nucleic acid addition during photo-mediated synthesis. UV light
removes the photolabile protecting group (circles) from the microarray surface, allowing the
addition of a single protected nucleotide to the growing oligonucleotide chain. The cycling of
DMD filtering, light deprotection, and nucleotide addition creates oligonucleotide features 60 to
100 bp in length on the NimbleGen microarray.

2. Contact printing (Patrick O Brown, Stanford University).

 VIKAS KUMAR SINGH

3. Non-Contact Printing (Pin and Ring, Bubble Jet, Ink Jet).

1. Noncontact inkjet printing technology delivers a small and accurate volume

(picoliters) of nucleotides to the first layer on the microarray surface.
2. Repeated rounds of base-by-base printing extend the length of specific
oligonucleotide probes.
3. Close-up of growing oligonucleotide chain with a base being added.
4. The final product is a 60-mer in situ-synthesized probe as a feature on a
microarray containing thousands of specifically synthesized probes.

Two-color vs. one-color detection,

Two-Color microarrays are typically hybridized with cDNA
prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are
labeled with two different fluorophores. Fluorescent dyes commonly used for cDNA labelling
include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the
green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm
(corresponding to the red part of the light spectrum). The two Cy-labelled cDNA samples are
mixed and hybridized to a single microarray that is then scanned in a microarray scanner to
visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined
wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to
identify up-regulated and down-regulated genes.
On the other hand in single channel microarrays or one
colour microarrays, the arrays are designated to give estimations of the absolute level of gene
expression. Therefore, a comparison of to conditions requires two separate single dye
hybridizations. As only a single dye is used in one colour microarrays.

Applications,
 Gene expression profiling.
 Discovery of drugs.
 Diagnostics and genetic engineering.
 in proteomics.
 Functional genomics.
 DNA sequencing.
 Toxicological research.

ADVANTAGES

 Provide data for thousands of genes.

 One experiment instead of many.
 Fast and easy to obtain results.
 Huge step closer to discovering cures for diseases and cancer.
 Different parts of DNA can be used to study gene expression.

DISADVANTAGE

 The biggest disadvantage of DNA chips is that they are expensive to create.
 The production of too many results at a time requires long time for analysis,
which is quite complex.
 The DNA chips do not have very long shelf life, which proves to be another
major disadvantage of the technology.
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Resolving different type of raised issues regarding reliability and reproducibility in DNA
microarray measurements.

Companies manufacturing them,

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References,
 http://www.gene-chips.com/
 http://www.bio.davidson.edu/courses/genomics/chip/chip.html
 http://www.cs.washington.edu/homes/jbuhler/research/array
 Dubey C. R, 2008, DNA Chips, A textbook for Biotechnology, S. Chand and
Company Ltd., New Delhi, 13th Edition, Pg. 194 – 197.
 www.gene-chips.com
 www.biotechnology4u.com
 www.biotechnologyforums.com
 www.ehow.com
 https://www.ncbi.nlm.nih.gov/pubmed/21901611
 DNA chips, microarrays and genomics P.K.Gupta, Joy K. Roy and Manoj Prasad.