CHAUDHARY CHARAN SINGH UNIVERSITY

MEERUT (U.P.)

Institute of Dental Studies & Technologies

Modinagar

M.D.S. (ORAL PATHOLOGY)

Seminar

“PCR & MICROARRAY”

Dr. Mohammad Ehtisham

 PCR & MICROARRAYS

The era of the pathologist relying entirely on the examination of tissue sections stained by
histochemical methods is gradually being replaced by advanced immunologic and molecular
techniques analysis of
• DNA
• RNA
• Protein structure or function
augment the process by which complicated infectious, inflammatory, metabolic and
neoplastic diseases are diagnosed and classified.

Molecular technology has become a crucial tool for identifying new genes with importance
in…
• Medicine
• Agriculture
• Animal production
• Health, environment
• Industry related to these areas.
Among the applications of molecular techniques is important to highlight the use of the
Polymerase Chain Reaction (PCR) in the identification and characterization of viral,
bacterial, parasitic and fungal agents.
It has been considered as an essential tool in molecular biology which allows amplification of
nucleic acid sequences (DNA and RNA) through repetitive cycles in vitro.
Mechanisms involved in this methodology are similar to those occurring in vivo during DNA
replication.

This technique was developed by Kary Mullis in the mid 80's.

"Beginning with a single molecule of the genetic material DNA, the PCR can generate 100
billion similar molecules in an afternoon.
The reaction is easy to execute.
It requires no more than a test tube, a few simple reagents, and a source of heat....."
Kary B. Mullis
(The inventor of PCR)
DEFINITION OF PCR
It is a genetic technique that occurs in vitro which allows the enzymatic synthesis of large
quantities (amplification) of a targeted region of DNA in exponential manner.
DNA is synthesized in the same manner as that seen in vivo (in the cells) using a DNA
polymerase (enzymes that cells use to replicate their DNA).

PRINCIPLE OF PCR
Polymerase chain reaction (PCR) is a powerful and widely used technique that has greatly
advanced our ability to analyze genes.
Genomic DNA present in cells contains many thousands of genes.
This makes it difficult to isolate and analyze any individual gene.
PCR allows specific DNA sequences, usually corresponding to genes or parts of genes, to be
copied from genomic DNA in a simple enzyme reaction.

Only requirement is that some of the DNA sequence at either end of the region to be copied
is known.
DNA corresponding to the sequence of interest is copied or amplified by PCR more than one
million fold and becomes the predominant DNA molecule in the reaction.
Sufficient DNA is obtained for detailed analysis or manipulation of the amplified gene.
ESSENTIAL COMPONENTS OF REACTION (PCR)

1. template DNA:
Template DNA containing genomic DNA sample from patient can be used in single or
double–stranded form.
Closed circular DNA templates are amplified slightly less efficiently than linear DNAs.

Ideally PCR requires only a single copy of target sequence as template.

2. Oligonucleotide Primers:
A pair of synthetic oligonucleotides should be short, single stranded, and complementary to
opposite strands of the flanking regions of the fragment of interest.
Standard reactions contain 0.1 - 0.5μM of each primer, which is sufficient for 30 cycles of
amplification of a 1-kb segment of DNA.
Higher concentrations of primer favour mispriming and may lead to nonspecific
amplification.
Oligonucleotide primers synthesized on an automated DNA synthesizer can be used for
standard PCR.

3. Thermostable DNA polymerase:

A thermostable DNA polymerase, which can withstand the denaturation temperatures (94-
95 ºC), is essential to catalyse the template dependent synthesis of DNA.
Originally the Klenow fragment of Escherichia coli pol I was used (Saiki et al., 1985).
This enzyme was inactivated at high temperatures required for separation of two strands of
DNA and hence had to be added freshly after every denaturation step.
This hurdle was overcome with the introduction of thermostable DNA polymerase,
Taq DNA polymerase isolated from thermophilic bacterium Thermus aquatics.
For a standard 25-50μl reaction, 0.5-0.25 units of Taq polymerase are used.

4. Deoxynucleoside triphoshates (dNTPs):

Standard PCRs contain equimolar concentrations of …
• dCTP, Deoxycytidine triphosphate
• dTTP, Deoxythymidine triphosphate
• dATP, Deoxyadenosine triphosphate
• dGTP, Deoxyguanosine triphosphate
(200-250 μM each).
These dNTPs are available commercially and supplied as pyrophosphate free mixtures.

4. Deoxynucleoside triphoshates (dNTPs):

dNTPs must be stored at -200 ºC.
During long term storage, small amounts of water evaporate and freeze over the walls of the
vial.
Hence in order to minimize the change in concentration, vials should be centrifuged before
use.

5. Divalent Cations:
Free divalent cations are needed for the activity of thermostable polymerases.
Usually Mg+2 is used.
Mg+2 binds to dNTPs and oligonucleotides.
Molar concentration of cation should exceed the molar concentration of phosphate groups in
the dNTPs and primers together.
Hence the optimal concentration of the cations should be calculated empirically for each
reaction.
A concentration of 1.5mM of Mg+2 is routinely used.
Excess Mg+2 will result in accumulation of non-specific amplification products and
insufficient Mg2+ will reduce the yield.

Buffer to maintain pH:

pH of reaction mixture is adjusted to 8.3 - 8.8 at room temperature for standard PCR.

Monovalent cations:
50 mM of KCl is used in standard PCR for amplification of DNA segments more than 500
base pairs in length.

Others:
Some researchers have reported that the efficiency of reaction is increased by inclusion of
10% dimethyl sulfoxide (DMSO) in the Taq polymerase buffer.
PROCEDURE
DNA or RNA suitable for PCR analysis can be obtained from most of tissue sources,
including…
• Fresh or frozen unfixed tissue,
• Cytology smears,
• Stained sections,
• Blood films and
• Body fluids like saliva also.
DNA extracted from formalin fixed paraffin wax embedded tissues can also be used for
amplification by PCR.
Such DNA is highly degraded as compared to that obtained from fresh or frozen samples
and cannot be used for analysis by techniques like southern blot analysis.
Fixatives such as Bouin’s solution should be avoided to fix tissues meant for PCR
amplification; whereas buffered formalin fixative can be used.
If the tissue remains in formalin for more than a few days, the extracted DNA may not be
successfully amplified.
Quality of DNA extracted from tissues embedded in paraffin wax is not affected even though
stored for long duration of time.
Amplification can be conveniently performed in a DNA thermal cycler.
The reaction mixture contains..
• A target sequence of 100-500 base pair length
• 50 mM KCl
• 10 mM Tris.HCI (pH of 8.4 at room temp)
• 1.5 mM MgCl2
• 100 μg/ml Gelatine
• 0.25 μM of each Primer
• 200 μM of each Deoxynucleotide Triphoshates
(dATP, dCTP, dGTP, and dTTP)
• 2.5' units of Taq Polymerase
Amplification of the target sequence is achieved by a repetitive series of cycles involving
three steps:
1. Denaturation of the template by heat.
2. Annealing of the oligonucleotide primers to single stranded target sequences.
3. Extension of the annealed primers by thermostable DNA polymerase.
DENATURATION
In PCRs catalysed by Taq polymerase, denaturation is carried out at 94-95 ºC, which is the
highest temperature the enzyme can withstand for 30 or more cycles without being
damaged.
During the first cycle, denaturation is carried out for 5 minutes to ensure complete
denaturation of the long molecules of template DNA.
But at times such longer duration of denaturation may be deleterious.
Denaturation for 45 seconds at a temperature of 94-95 ºC is recommended for routine
amplification of linear DNA templates containing 55% or lesser amount of G + C.
Higher temperatures may be required to denaturate templates containing higher amounts of
G + C.

In addition, longer the DNA templates, greater is the denaturation time required.
If denaturation temperature is too low or if time is too short, only the A – T rich regions of
template DNA will be denatured.
Such DNA will re-anneal back when denaturation temperature is reduced later during PCR
cycle.

ANNEALING
Annealing temperatures range from 55-65 ºC depending on the primer sequence and length.

Annealing temperature is critical.

If annealing temperature is too high,
the oligonucleotide primers anneal poorly and the amplified DNA is too low.
If annealing temperature is too low,
Nonspecific annealing of primers may occur, resulting in the amplification of unwanted
segments of DNA.
Annealing temperature can be optimized
by performing series of trial PCRs at room temperatures ranging from 2-100 ºC below the
melting temperatures of oligonucleotide primers.
Also a sequential series of annealing temperatures can be used in a routine PCR.

EXTENSION
DNA polymerase catalyses the extension of oligonucleotide primers and there by a new
strand, having sequences complementary to template strand is synthesised.
Optimal temperature for DNA synthesis may vary slightly depending on the DNA
polymerase used.
When Taq polymerase is used, the ideal temperature for DNA synthesis is about 72-78 ºC.
Taq polymerase can insert about 2000 nucleotides every minute at this temperature.
Extension of from one primer proceeds beyond the sequence complementary to the binding
site of the other primer during the first two cycles.
In the next cycle, the length of the DNA molecules produced is equal to the segment of DNA
delimited by binding sites of primers.
From the third cycle onwards, this segment of DNA is amplified geometrically while the
longer amplification products accumulate arithmetically.
NUMBER OF CYCLES
Number of cycles needed for amplification depends on…
• Number of template DNA sequences present in the reaction mixture
• Efficiency of primer extension.
Amplified products accumulate exponentially.
PCR PHASES
A basic PCR run can be broken up into three phases:
1. EXPONENTIAL:
Exact doubling of product is accumulating at every cycle (assuming 100% reaction
efficiency).
Reaction is very specific and precise.

PCR PHASES

PCR PHASES
2. LINEAR:
(HIGH VARIABILITY):
Reaction components are being consumed, the reaction is slowing, and products are starting
to degrade.
PCR PHASES
3. PLATEAU:
(END-POINT: GEL DETECTION FOR TRADITIONAL METHODS):
Reaction has stopped, no more products are being made and if left long enough, the PCR
products will begin to degrade.
In most cases the plateau is unavoidable but by the time it occurs, adequate amounts of
product will have accumulated.
If more material is required, multiple reactions can be easily set up.
ANALYSIS OF THE PCR PRODUCTS
BASIC ANALYSIS
Basic analysis of PCR products is based on the fact that double stranded DNA molecules
migrate through the gels on the basis of their size; larger products migrate more slowly than
smaller ones.
Simplest methods of analysing the PCR products are
• Agarose gel electrophoresis
• Polyacrylamide gel electrophoresis
Followed by Ethidium bromide staining and viewing under UV illumination.

Schematic illustration of a typical horizontal gel electrophoresis setup for the separation of
nucleic acids.

AGAROSE GEL ELECTROPHORESIS METHOD

GEL ELECTROPHORESIS BASED IMAGE ANALYSIS
• Agarose gels, stained by Ethidium bromide (A)
• UV light (B)

Choice of the gel depends on the size and resolution required.

For larger products (300 bp and larger) agarose gels are more convenient and use less toxic
reagents.
If smaller fragments must be accurately sized, then polyacrylamide gels are required.

SINGLE-STRANDED CONFORMATIONAL POLYMORPHISM ANALYSIS (SSCP)

If PCR products are run when they are single stranded under non denaturing conditions,
their rate of migration is determined by their primary sequence as a result of sequence-
specific self-folding.
This is determined by the amount and type of complementary sequence binding within the
DNA strand.
A single base change results in altered mobility of products in the majority of cases.

SINGLE-STRANDED CONFORMATIONAL POLYMORPHISM ANALYSIS (SSCP)

This method is known a single-stranded conformational polymorphism (SSCP) analysis.
It is a very powerful method for screening for mutations in gene fragments of interest.
Products can be denatured and encouraged to remain single-stranded using buffer systems
high in concentration of formamide and by running them on non-denaturing
polyacrylamide gels at low temperatures (for example, 5 ºC).
HETERODUPLEX ANALYSIS
In this method heterogeneous PCR products are subjected to heat denaturation followed by
rapid cooling on ice, which encourages the formation of mismatched products rather than
perfectly matched ones.
Any mismatched ‘heteroduplexes’ can be identified by altered electrophoretic mobility.
Heteroduplex analysis can be used to search for point mutations.
SEQUENCE ANALYSIS
Sequence analysis is a rapid, inexpensive and reliable method, where commercially-available
user-friendly kits are used.
Generation of a high-quality template is essential for sequencing.
DNA extracts from paraffin-wax-embedded samples is limited but fragments of up to 300 base
pairs can be sequenced directly without cloning of PCR products.

ADVANTAGES
SIMPLE TECHNIQUE
PCR is a relatively simple technique that can detect a nucleic acid fragment and amplify this
sequence.
SENSITIVITY
This technique offers sensitivity because from small amounts of genetic material can be detected
target sequences in a sample.

SPECIFICITY
Also this offers specificity due to a specific sequence of DNA is amplified through strict
conditions.
FAST TECHNIQUE
It is considered a fast technique compared with other methods to detect microorganisms such as
bacteria, fungus or virus, which require isolation and culture using culture media or cell lines.
VERSATILITY
Versatility due to the genetic sequences from various microorganisms can be identified with the
same reaction conditions for diagnosis of different pathologies

VARIATIONS ON THE BASIC PCR

Real-Time PCR
A real-time PCR is used to amplify and simultaneously detect or quantify a targeted DNA
molecule.
Real-Time chemistries allow for the detection of PCR amplification during the early phases
of the reaction.
Traditional methods use Agarose gels for detection of PCR amplification at the final phase
or end-point of the PCR reaction.
Gel electrophoresis is often used to measure the size of the amplicons.
Real-Time PCR
This method is both inexpensive and simple.
But, size analysis has limited specificity since different molecules of approximately the same
molecular weight cannot be distinguished.
Hence, gel electrophoresis alone is not a sufficient PCR end-point in many instances.
Real-time PCR overcomes this limitation.
The reaction is placed in to a real-time PCR machine that watches the reaction occur with a
camera or detector.

There are two general approaches used to obtain a fluorescent signal from the synthesis of product
in PCR.
• First depends upon the property of fluorescent dyes such as SYBR Green I to bind to
double stranded DNA and undergo conformational change that result in an increase in
their fluorescence.
• Second approach is to use fluorescent resonance energy transfer (FRET).

Multiplex PCR (M-PCR)

• Multiplex PCR (M-PCR) is the simultaneous detection of more than one target
sequence.
• M-PCR is the simultaneous amplification of more than one target sequence in a single
reaction tube using more than one primer pair.
 • Such co-amplification reactions where in two or more targets are amplified in a single
reaction is dependent on the compatibility of the PCR primers used in the reaction.

All primers in the reaction must have similar melting temperatures (Tm)
So they anneal to and dissociate from DNA sequences at approximately the same
temperatures,
Allowing both amplifications to proceed simultaneously at the selected temperature.

Multiplex PCR (M-PCR)

Each amplification proceeds independently of the others and each specific amplification product
is synthesized independently.
Primers should also be chosen such that the target sequences are of approximately the same size
range, so that each of the target sequences is synthesized efficiently and at equal rates.

M-PCR has been used for detection of many HPV types.

Two different blood borne viruses, human immunodeficiency virus type 1 and hepatitis C virus,
have been detected by M-PCR.
Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) have been detected in preserved paraffin
sections of lung tissue from immunocompromised patients.

The advantage of M-PCR…

• Ability to detect more than one agent in a single test.
• To analyze specimens such as respiratory tract secretions, from which several different
viruses can be recovered, M-PCR is a cost effective technique.
• Another advantage of M-PCR is its high degree of sensitivity.

Drawbacks of M-PCR are few and these include…

• Cost factor,
• Considerable time required to develop and evaluate new assays
• Need for effective anti-contamination measures.

Nested PCR
• This PCR increases the sensitivity due to small amounts of the target are detected by using
two sets of primers, involving a double process of amplification.
• First set of primers allows a first amplification.
• Product of this PCR is subjected to a second PCR using the second set of primers.
• These primers used in the second PCR are specific to an internal amplified sequence in the
first PCR.
Therefore, specificity of the first PCR product is verified with the second one.
Disadvantage of this technique is…
Probability of contamination during transfer from the first amplified product into the tube in
which the second amplification will be performed.
Contamination can be controlled using primers designed to anneal at different temperatures.
Contamination can also be controlled by adding ultra-pure oil to make a physical separation of
two mixtures of amplification.

In-cell PCR
All the in-cell PCR techniques attempt to create double-stranded or single stranded DNA
amplicon within the cell.
First step involves denaturation of double-stranded DNA (dsDNA) to single stranded DNA
(ssDNA), if target sequence is DNA.
For RNA target-specific amplification, the RNA template is already single stranded and reverse
transcription is carried out to create a DNA template.

Primers are then annealed to the respective ends of the desired target sequence and a thermostable
enzyme is used to extend the correctly positioned primers.
Subsequent rounds of thermocycling increase the copy number of the desired target sequence.
Once the amplicon is created, detection is carried out using a labelled primer or nucleotide which
can be demonstrated directly by immunocytochemical techniques.

Several In-cells PCR techniques are now described, these include

• DNA in-situ PCR
• Labelled primer driven in-situ amplification (LPDISA)
• PCR ISH
• Reverse transcriptase in-situ PCR (RT IS-PCR)
• In-situ PNA PCR (IS-PNA-PCR) and PCR-PNA-ISH
• In-situ Immuno-PCR

RNA PCR
RNA can also be used as template for PCR following reverse transcription.
This technique is useful for the study of expressed gene sequences and retroviruses.
In many cases the PCR primers can be designed to specifically amplify the DNA sequences by
choosing fragments spanning intronic regions.

Basis of reverse transcriptase PCR is the conversion of RNA to DNA.

Such DNA is termed complementary DNA (cDNA) and is transcribed from the RNA by the
enzyme reverse transcriptase.
cDNA can now be used as a template for PCR.
Detection and quantification of mRNA in cells has been done successfully by using reverse
transcriptase PCR.

RNA PCR
Total amount of mRNA in cells may be as less as 2%, but its analysis is important since mRNA
provides a direct measure of the amount of transcription in cell.
An important application of reverse transcript PCR is the detection and quantification of mRNA
transcribed from tumour associated translocations.
Translocation of gene segments is common in many neoplasms especially hematopoietic
malignancies.

APPLICATIONS OF PCR
Detecting pathogens using genome-specific primer pairs in clinical samples –
All organisms have DNA sequences (rDNA) which code for ribosomal RNA.
There are regions in the rDNA which vary between genera and species. These variable
regions are amplified and then sequenced to determine the identity of the unknown
organisms
Detection of viral pathogens and other micro-organisms which persist in low levels in infected
cells and are difficult to be identified by routine methods.

Quantitative Real-Time can be used to detect viral genomes such as HIV or HPV.
Diagnosis of genetic disorders such as phenylketonuria, haemophilia, sickle cell anaemia,
thalassemia.
Identification of genetic mutations like deletions, insertions and point mutations.
Screening specific genes for unknown mutations.

Identification and analysis of mutations in eukaryotic DNA.

Gene polymorphisms
Gene expression
 MICROARRAYS
Term microarray was first introduced by Schena et al. in 1995.
First genome of an eukaryotic species completely investigated (saccharomyces
cerevisiae) by a microarray was published in 1997 (lashkari et al., 1997).
 Gene: Hereditary DNA sequence at a specific location on chromosome
 Genome: One complete set of genes in an organism (a haploid set).
 Genomics: Study of organisms in terms of their genome.
 Gene expression: It is the term used to describe transcription of information
contained within DNA into mRNA that are translated to proteins .
 Transcriptome: All the messenger RNA (mRNA) molecules transcribed
from the genome.
 Hybridization: It is the specific reassociation of complementary strands of
nucleic acids.
 Library: A collection of cloned fragments that represents entire genome
- Genomic library(Genomic DNA) : Both introns and exons are represented
-cDNA library- Only exons are represented
DEFINITION
Microarrays are miniaturized biological devices consisting in molecules, for
example DNA or protein, named the "probes", that are orderly arranged at a
microscopic scale onto a solid support such as a membrane or a glass microscope
slide.

• Each microarray contains thousands of genes.

MICROARRAY
A microarray is typically a glass slide on to which DNA molecules are fixed in an
orderly manner at specific locations called spots (or features).
A microarray may contain thousands of spots and each spot may contain a few
million copies of identical DNA molecules that uniquely correspond to a gene.
DNA in a spot may either be genomic DNA or short stretch of oligonucleotide
strands that correspond to a gene.
Spots are printed on to the glass slide by a robot or are synthesised by the process of
photolithography.
A microarray may contain thousands of ʻSPOTSʼ.
Each spot contains many copies of the same DNA sequence that uniquely represents
a gene from an organism.
Spots are arranged in an orderly fashion into Pen-groups

MICROARRAYS MEASURE EVENTS IN GENOME

An event may be the…
• Transcription of a gene
• Binding of a protein to a segment of the DNA,
• Presence or absence of a mutation,
• A change in the copy number of a locus,
• A change in the methylation state of the DNA,
• Any of a number of states or activities that are associated with DNA or RNA molecules.
As a genomic readout, microarrays identify where these events occur.
PURPOSE & MECHANISM
Purpose of a microarray is to examine expression of multiple genes simultaneously in response to
some biological perturbation.
More generally, a microarray serves to interrogate the concentrations of molecules in a complex
mixture and thus can serve as a powerful analytical tool for many kinds of experiments.
To understand how this occurs, it may be useful to review the structure of DNA and examine how
the unique structure of this molecule plays a role in identifying itself.

DNA is made up of four chemical building blocks called bases: adenine, cytosine, guanosine, and
thymidine (A, C, G, or T).
As individual subunits these building blocks are also referred to as nucleotides.
A strand of DNA consists of a sugar phosphate backbone to which these bases are covalently
linked such that they form a series.

Because these four bases can form sequences, it is possible to use them to encode information
based on their patterns of occurrence.
Indeed, from an information point of view, DNA has a potential data density of 145 million bits
per inch and has been considered as a substrate for computation.
Amount of DNA, and thus the amount of sequence, varies from organism to organism.
For instance,
Microorganism Escherichia coli has 4.5 million bases of sequence, whereas human cells have
about 3 billion bases.
Exactly how much biological information is encoded in these sequences is unknown, representing
one of the deepest mysteries of biology, but microarrays provide a way to gain clues.
Bases of one strand interact with the bases of the other strand according to a set of pairing rules,
such that
• A pairs with T
• C pairs with G.
Thus, if one knows the sequence of one strand, by definition, one then knows the sequence of the
opposite strand.
This property has profound consequences in the study of biology.
It is also what the cell uses to replicate itself.
As the interaction between the bases is non-covalent, consisting only of hydrogen bonds,
Strands can essentially be melted apart and separated,
Thus opening the way for a copying mechanism to read each single strand and re-create the
second complementary strand for each half of the pair, resulting in a new double-stranded
molecule for each cell.
This is also the mechanism by which cells express genes.
Strands are opened by the gene expression machinery so that some number of RNA copies
of a gene can be synthesized.
RNA transcript has the same sequence as the gene with the exception that uracil (U)
replaces T, though the hybridization pairing rules remain the same (U and T can both pair
with A).
This property of complementarity is also what is used for measuring gene expression on
microarrays.
Just as energy can melt strands apart and separate them into single molecules, the process is
reversible such that single strands that are complementary to each other can come together
and re-anneal to form a double stranded complex.
This process is called hybridization.
It is the basis for many assays or experiments in molecular biology.
In the cell, hybridization is at the centre of several biological processes, whereas in the lab
complementarity is identity and thus hybridization is at the centre of many in vitro reactions
and analytical techniques.
Molecules can come from completely different sources, but if they match, they will hybridize.
Types of Microarray-

 DNA Microarray
 Protein Microarray
 Tissue Microarray
 Chemical compound Microarray

WHAT IS DNA MICROARRAY?

It is a collection of microscopic DNA spots commonly representing single genes , arrayed on
a solid surface by covalent attachment to chemically suitable matrices.
• Solid support-
• Plastic,
• Glass or
• silicon chip

 DNA chips

 Biochips

 Gene chips

 Gene arrays

 Genome chips

 Genome arrays

How do Gene arrays work?

The whole process is based on hybridization.
It works by exploiting the ability of given mRNA molecule to bind specifically to or
hybridize to the corresponding DNA template.
THREE important things:
1. Solid support (Glass slide)
2. Probe
3. Target

For Example
One common use of DNA microarray is which gene is activated or repressed when two
population of cells are compared.
Lets compare what happens to yeast genes when grown in aerobic versus anaerobic
conditions.

• During this process genes are activated or suppressed in order to survive

• Next spin the tubes , Yeast settles down as pellet at the bottom. Remove the
supernatant liquid and extract mRNA
• Make cDNA and label with green and red dye – yeast grown in aerobic condition are
labelled green and anaerobic red.
STEPS OF MICROARRAY BASED ANALYSIS

1. Probe preparation and Microarraying

2. Target Preparation
3. Hybridization
4. Microarray image acquisition and data analysis

OLIGONUCLEOTIDE ARRAY
• DNA Arrays are composed of probes
• Each probe is a sequence of 25 nucleotides
OLIGONUCLEOTIDE ARRAYS
(trademarked as a GeneChip by Affymetrix)
Affymetrix GeneChips are the most ubiquitous and long-standing commercial array
platform in use.
The arrays consist of 25 oligonucleotides synthesized in situ on the surface of a glass chip.

It is generally used for one of two things:

Quantification of the amount of mRNA in a single sample (e.g. to determine the amount of
mutated vs. non-mutated mRNA)

Comparison of two different samples hybridized to two separate arrays.

Probes are larger pieces of DNA
Generated from commercially available cDNA library or
PCR used to amplify specific genes from genomic DNA.

MICROARRAY FABRICATION
Covalent immobilization on either glass or polypropylene surfaces.
Example
 Treatment of glass slides with silane allows amino covered glass to bind amino linked
probes.
 Coating with polylysine allows direct charge couple binding of ployanionic cDNA
probes.
 UV photo crosslinking adds covalent bond.

• Green labelled cDNA and red labelled ones are mixed together (call the target) and
put on the matrix of spotted single strand DNA (call the probe).
• The chip is then incubated one night at 60 degrees.
• At this temperature, a DNA strand that encounter the complementary strand and
match together to create a double strand DNA.
MICROARRAY IMAGE ACQUISITION
 Readout of the microarray is captured as a colour coded image using a dual-laser scanner
for fluorescence detection.
 Scanner excites the fluorochromes associated with the target-probe DNA complex and
measures fluorescence emission intensities at each DNA spot by scanning the microarray
surface.
 Special software computes the intensities of the two fluorochromes at each location.

PROTEIN MICROARRAY
• It is a piece of glass on which different molecules of proteins have been affixed at separate
locations in ordered manner.
• Identification of protein-protein interactions, protein-phospholipid interactions, small
molecule targets, and substrates of proteins kinases. They can also be used for clinical
diagnostics and monitoring disease states .
 • Most common protein microarray is antibody microarray, where antibodies are spotted
onto the protein chip and are used as capture molecules to detect proteins.
• Proteins can be externally synthesized & attached to array or synthesized in-situ
• Biosynthesis or chemical synthesis
Types of capture molecules..
 Most common- Antibody
 Others- Nucleic acid, receptors, enzymes, proteins
Preferred method of detection currently is fluorescence detection.
Fluorescent detection is safe, sensitive, and can have a high resolution.

APPLICATIONS OF PROTEIN CHIPS

 Biochemistries of thousands of proteins can be characterized and quantified.
 Discover new functionalities for previously uncharacterized proteins.
 Proteome chips have been used to study protein-protein interactions, Protein-DNA
interactions, Protein-lipid interactions Protein-drug interactions, Protein-receptor
interactions and antigen-antibody interactions.
 Proteome chips have been used to study kinase activities.

TISSUE MICROARRAY
DEFINITION:
• It is an approach of simultaneous screening of large collectives of tissue specimens for any
molecular alteration by relocating tissues from conventional paraffin blocks, such that
tissues from multiple patients could be seen on same slide.
• Tissue microarrays (TMAs) are used to analyze the expression of genes simultaneously in
multiple individual tissue samples on one slide.
HISTORY
• Multi-tissue blocks -first introduced by H. Battifora in 1986 with his so called “multitumor
tissue block" and modified in 1990 with its improvement, "the checkerboard tissue block" .
• In 1998 J. Kononen and collaborators developed the current technique, which uses a
novel sampling approach to produce tissues of regular size and shape that can be more
densely and precisely arrayed.

TISSUE MICROARRAY SYNTHESIS

• Tissue Microarrays are produced using a microtome which sections tissue into 4 - 5
micrometer sections.
 • These core tissue biopsy sections are taken from specific areas of interest from paraffin-
embedded tissue blocks.
• These cylindrical cores of tissue are re-embedded into an arrayed blank recipient blocks.
Using this method

TYPES OF TISSUE MICROARRAYS:

CRYO - TISSUE MICROARRAYS:
Uses frozen tissues, due to their freezing are superior to formalin fixed tissues for RNA and
protein analysis.
Also, antibodies work much better with frozen tissues.

MULTI-TUMORS TISSUE MICROARRAYS:

have many different types of tissues aligned on the slide.
PROGRESSION TISSUE MICROARRAYS:
This type of tissue microarray examines different stages of tumor (or disease) progression within a
given organ.
For example: examination of tumors in the breast.
These slides can then be assayed for markers of interest, or biochemical analysis of the samples
can be done.

PROGNOSIS TISSUE MICROARRAYS:

Disease samples such as tumor biopsies can be taken from patients and examined.
These samples can be used for clinical follow-ups to monitor the patient's progression.
Data is then analyzed and compared with other clinical data

CHEMICAL COMPOUND MICROARRAY

• A Chemical compound microarray is a collection of organic chemical compounds
spotted on a solid surface, such as glass and plastic.
• In chemical genetics research, they are routinely used for searching proteins that binds
with specific chemical compounds, and in general drug discovery research.
• They are used for searching potential drugs for therapeutic targets.

BIBLIOGRAPHY
• Cellular Pathology Technique Fourth edition by C. F. A Cullings.
• Theory & practice of histological techniques by John D Bancroft.