Unit 13 MolecularTechniques

UNIT 13
MOLECULAR TECHNIQUES

Structure
13.1 Introduction 13.6 Hybridoma and Monoclonal
Antibodies
Objectives
13.7 ELISA and Immunodetection
13.2 Northern, Southern, and
Western Blotting 13.8 Summary

13.3 DNA Fingerprinting 13.9 Terminal Questions

13.4 Molecular DNA Markers 13.10 Answers

RAPD, RFLP, SNPs

13.5 DNA sequencing, PCR, and

Reverse Transcriptase-PCR

13.1 INTRODUCTION
Earlier units have introduced you to the basics of biotechnology, emphasizing
various plant tissue culture methods. You have also read about applications of
plant tissue culture, such as micropropagation, androgenesis, gynogenesis,
embryo culture and germplasm conservation. The present unit provides
information about molecular biology and plant biotechnology techniques such
as Southern, Northern, and Western blotting used to identify DNA, RNA and
proteins. In addition, DNA amplification techniques, DNA markers, and
hybridoma technology have been dealt with in detail.

Objectives
Objectives
After studying this unit, you would be able to:

describe the techniques of Southern, Northern, and Western blotting;

know about the ELISA technique;

describe different types of DNA markers;

appreciate the details of the DNA fingerprinting technique; and

describe hybridoma technology. 87

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13.2 NORTHERN, SOUTHERN, AND WESTERN
BLOTTING
The mixture of DNA, RNA and protein fragments can be separated by gel
electrophoresis. Further, the separated gel bands can be stained with specific
dyes and studied. The identity of these bands can be confirmed by using a
known molecular probe that illustrates/indicates the similarity to one or more
bands. The separated bands can be hybridized using probes within the gel.
Hybridization using probes is carried out after transferring the bands to the
nitrocellulose membrane. Blotting refers to transferring bands onto a
membrane and then hybridizing them with a specific probe. In a sample,
Southern blotting detects specific DNA sequences, Northern blotting detects
specific RNA sequences, and Western blotting detects specific protein
sequences. As a result, the main distinction between the three blotting
techniques is the macromolecule type that they detect.

Southern Blotting

The technique was named after a biologist Edward M. Southern who

developed this technique in 1975. Southern blot hybridization refers to
detecting specific DNA fragments that have been separated by gel
electrophoresis (Fig. 1). The DNA segments are first digested with a restriction
enzyme, and the digested sample is subjected to electrophoresis. After that,
the separated DNA fragments in the gel are denatured in the buffer with the
help of an alkali solution. The single-stranded fragments from the gel get
transferred to the surface of the nitrocellulose membrane. The bands firmly
adhere to the paper based on the affinity of DNA for nitrocellulose paper.

The gel is laid on a buffer-saturated filter paper placed on a solid support. The
two edges of the gel are immersed in the buffer. A nitrocellulose membrane is
placed on top of the gel, which is then topped with a stack of papers. A weight
of about 1 kg is placed on the top of the stack. The buffer solution is drawn by
the filter paper wick, then through the gel to the nitrocellulose membrane, and
finally through the layer of paper. As the buffer passes through the gel, it
carries single-stranded DNA, which binds to the nitrocellulose membrane. The
sheet is exposed to the probe under conditions favoring hybridization. The set
is left up for several hours or overnight (Fig. 13.1). The paper towels are
discarded, and the nitrocellulose membrane has single-stranded DNA bands
that get blotted onto it. The membrane is baked at 80oC for 2 to 3 h to fix the
DNA permanently on the membrane. The membrane with DNA bands can
hybridize with labelled DNA or RNA probe (32P) (Fig. 13.1). A probe is a
labelled DNA sequence that hybridizes with one or more fragments of the
digested DNA sample after they were separated by gel electrophoresis, thus
revealing a unique blotting pattern characteristic of a specific genotype at a
specific locus. The nitrocellulose sheet containing the bound single-stranded
DNA fragments is placed in a sealed box along with the buffer containing
labelled DNA probe specific for the target DNA sequence. After the
hybridization, the sheet is washed thoroughly to remove unhybridized probes
(unbound DNA). The sheet is viewed using autoradiography or ultraviolet light
depending on the labels used (radioactive or fluorescent). Autoradiographs are
88 obtained by exposing the X-ray film to the hybridized membrane.
Unit 13 MolecularTechniques

Fig. 13.1 Diagram showing set up in the Southern blotting.

Southern blotting is used to detect specific DNA fragments of varying sizes.

The technique is used in DNA profiling or DNA fingerprinting. The techniques
are helpful in the discovery of genes located on a particular DNA sequence or
gene within the entire genome. It is used as a confirmatory test to ensure that
a particular DNA segment has been successfully incorporated into the genome
of another organism and also helps in assessing the evolutionary relationship
among organisms. The Southern blotting can also be used to determine the
genetic alterations causing a mutation due to structural change in the gene
controlling the trait of interest.

Northern blotting

Northern blotting is a method to detect specific RNA molecules from a mixture

of RNA. The technique is used to analyze RNA samples from a particular
tissue or cell type to measure the RNA expression of a particular gene. The
technique was developed by James Alwine, David Kemp, and George Stark
at Stanford University in 1977. The mRNA bands are transferred from the gel
to the nitrocellulose membrane or diazotized amino benzyloxy methyl paper.
The mRNA bands blotted on the nitrocellulose membrane can be hybridized
with labelled DNA or RNA probes. The RNA samples are then loaded onto an
agarose gel an electric current is passed through the gel, causing the RNA in
each sample to reach the bottom. The distance moved depends on the size of
the RNA fragment. The RNA molecules are separated according to their sizes
using gel electrophoresis. Small RNAs move faster while larger RNAs migrate
slowly. The RNA within the sample is denatured to get single strands,
formaldehyde is used as a denaturant.

After separation, the RNA is transferred from the gel onto a blotting
membrane. Each RNA molecule retains its position relative to all the other
molecules, a probe, which is a small piece of DNA or RNA with a
complementary sequence to a specific RNA sequence in the sample, is used
to treat the nitrocellulose membrane. It allows the probe to hybridize, or bind,
to a specific RNA fragment on the membrane (Fig.13.2). Next, the probe is
radioactively labelled or using a fluorescent dye (Fig. 13.2). The filter is then 89
Block 4 Biotechnology - Techniques and Applications
placed on a film for autoradiography, and finally, the film will be developed.
The probes can be seen on the autoradiograph if paired with a specific region
of RNA on the filter. The gene gets expressed shows a band that will be visible
in the column containing the tissue or cells only.

The technique of Northern blotting is helpful for the study of gene expression.
The position of bands on the blot provides size RNA. The Northern blot
analysis of RNA samples from different cells or tissues enables researchers to
determine the expression of the gene along with the relative levels of
expression in cells. Hence the technique helps to identify where and when a
specific section of DNA of a known sequence gets transcribed. This technique
also detects gene variants and identifies closely related species.

Fig. 13.2: Diagram showing set up in the Northern blotting.

Western blotting
Towbin and Julian Gordon (Friedrich Miescher Institute) and Theophil
Staehelin (at Hoffmann-La Roche) introduced western blotting in 1979. It is a
rapid and sensitive assay for detecting and characterizing proteins. It is based
on the ability of a specific antibody to bind a given protein or peptide in a
mixture of is used to detect its antigen-specific. It is based on the
immunochromatography principle, in which proteins are separated into
polyacrylamide gel based on their molecular weight. The proteins are
separated using polyacrylamide gel electrophoresis and then transferred to the
nitrocellulose membrane to which they bind. The proteins are detected using a
specific primary antibody, and secondary enzyme labelled antibody and
substrate. The membrane is used for probing with a specific labelled antibody.
The nitrocellulose membrane is first incubated with a primary antibody to the
protein of interest. The antibody binds to the protein, and an antibody-protein
complex is formed. Next, the nitrocellulose paper is incubated with a
secondary antibody, and it specifically binds to the primary antibody, finally, a
secondary antibody tagged with an enzyme helps detect and visualize the
bound antibody. For enzymatic reaction, the substrate is added, and the
reaction mixture is allowed to incubate. The colored bands can be noted at
sites where the primary antibody-secondary antibody-enzyme complex is
90 formed (Fig.13.3).
Unit 13 MolecularTechniques
If the radiolabelled antibody is used, the signal is detected with the help of
autoradiography. The target protein is marked using a primary and secondary
antibody for visualization. The unbound antibody is washed off, leaving only
the bound antibody to the protein of interest. The bound antibodies are then
detected by developing the film. As the antibodies only bind to the protein of
interest, only one band should be visible. The thickness of the band
corresponds to the amount of protein present. The technique detects proteins
of a particular specificity and determines the size and amount of protein in a
given sample. For example, the western blotting technique is the confirmatory
test for HIV that detects anti-HIV antibodies in the patient's serum and detects
defective proteins.

Fig.13.3: Various steps involved in Western blot.

SAQ 1
Match the items in column A with those in Column B.

Column A Column B

a) Detection of DNA 1. Western blots

fragments
b) Detection of RNA 2. Southern blots
molecules
c) Towbin & Julian Gordon 3. Northern blots

d) Helps in the identification of 4. Molecular probe

specific bands

13.3 DNA FINGERPRINTING

The technique of DNA fingerprinting was developed in 1984 by British
geneticist Alec Jeffreys, who discovered that certain sequences of highly
variable DNA (known as minisatellites), do not contribute to the functions of
genes, are repeated within genes. In most cases, minisatellites are defined as
the repeated in the tandem repetition of a short (6 to 100 bp) motif that spans
0.5 kb to several kilobases in length. They show polymorphism, which results
from variations in the number of repeats. They can cross-hybridize with tens of
other similar loci throughout the genome. Hence, they are used in DNA
fingerprinting to identify an individual. DNA fingerprinting is used to isolate and
identify variable elements within DNA sequences. 91
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DNA is extracted from various places, including skin, hair, or blood cells
(WBCs). The DNA is purified and cut at specific points along the strand with
restriction enzymes. Specific restriction endonucleases digest the DNA. The
fragments produced of varying lengths are placed on the gel and then
subjected to electrophoresis. The DNA fragments are separated based on
length (Fig.13.4). The shorter the fragment, the more quickly it moves toward
the positive pole (anode). It is based on restriction fragment length
polymorphism (RFLP). The shorted double-stranded DNA fragments are then
subjected to a blotting technique in which they were split into single strands
and transferred to a nylon membrane. The fragments are exposed to DNA
probes. Fragments of DNA are made radioactive and made to bind to
minisatellites. The DNA strands are subjected to autoradiography. A piece of
X-ray film was then exposed to the fragments, and a dark mark was produced
at any point where a radioactive probe had become attached.

The location of the DNA fragments containing specific DNA sequences in the
gel is determined using a labelled probe having complementary sequences.
The DNA fragments that bind these probes show variable lengths from one
person to the other because of a variable number of short tandem repeats
(STR) in the genome. Generally, 13 STR, which is supposed to be
polymorphic, are analyzed. The amount of polymorphism generated is used to
identify the individual or specimens.

Fig. 13.4: Various steps involved in DNA fingerprinting technique.

13.4 MOLECULAR DNA MARKERS (RAPD,

RFLP, SNPs)
Molecular markers are DNA sequences that are used in molecular biology and
biotechnology to distinguish one particular sequence of DNA from another in a
pool of unidentified DNA. The DNA sequences provide many visual markers
that can be used to map their locations in the chromosome of various species.
The molecular markers that are extensively used in the preparation of genome
92 maps include RFLP, RAPD, and SNPs.
Unit 13 MolecularTechniques
Restriction Fragment Length Polymorphism (RFLPs)
The technique detects alterations in specific regions in the chromosome of
different strains/species. The technique was developed by Botstein and
coworkers in 1980. It is noted whether or not a recognition site for the same
restriction endonuclease exists in the same region of a chromosome from a
different individual of a species. The variation appears in the form of bonds. An
RFLP map depicts the location of different DNA sequences used as probes
(Fig.13.5). The enzyme treatment produces fragments of different lengths for
the same chromosome region of different individuals. Gel electrophoresis
detects the differences, generally followed by hybridization using a labelled
probe specific for the chromosome region. The DNA sequences used as a
probe have features like a strong hybridization signal, produce small bands,
and show polymorphism in different species strains.

DNA is isolated separately from the parent strains, the progeny, i.e. F1 and F2
plants, are used to determine RFLPs with various probes showing
hybridization. DNA is digested with a restriction enzyme, and the product
obtained is subjected to electrophoresis and Southern hybridization using
different probes. Short, single- or low-copy genomic DNA or cDNA clones are
typically used as RFLP probes.

The probes are used to screen different or related species, few species show
polymorphism for several probes. Instead, probes reveal a single band that
shows the difference in mobility in the two parental strains. One probe can
detect a slow-moving band, while the other probe can detect a fast-moving
band. The distance between the two markers can be measured to prepare a
map. This map depicts the relative positions of the chromosome.

We can detect the loci affecting the trait by mapping using RFLP markers.
These markers are specific to a single clone/restriction enzyme combination.
Most RFLP markers are co-dominant (both alleles in the heterozygous sample
will be detected) and highly locus-specific. The RFLP probes are frequently
used in genome mapping and in variation analysis (genotyping, forensics,
paternity tests, genetic disease diagnostics, and many more).

Fig. 13.5: Steps involved in RFLP.

Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA (RAPD) is a PCR based technique for
identifying genetic variation. RAPDs are generated using random sequences 93
Block 4 Biotechnology - Techniques and Applications
generally 10 oligonucleotides long. These markers are DNA fragments
generated from PCR amplification of random segments of genomic DNA with
a single primer of the arbitrary nucleotide sequence. RAPD markers are used
as primers for PCR amplification of genomic DNAs. They act as dominant
markers and are recognized by the primer used for amplification. The RAPD
technology was developed by Williams and his coworkers in 1990. They
used short synthetic oligonucleotides (10 bases long) of random sequences as
primers to amplify nanogram amounts of total genomic DNA under low
annealing temperatures by PCR.

RAPD involves using a single arbitrary primer in a PCR reaction, resulting in

the amplification of many discrete DNA products. The target DNA sequence is
amplified with the help of primers, DNA polymerase, di-deoxynucleotide tri-
phosphates, magnesium and reaction buffer. The DNA is made single-
stranded by raising the temperature to 94° C (denaturation) followed by
annealing primer to their target sequences on the template DNA at a lower
temperature (about 40 to 65° C). The temperature at which the thermostable
Taq DNA polymerase activity is optimal (i.e., usually 72°C) is chosen. The
polymerase extends the DNA-primer hybrids' 3' ends toward the other primer
binding site. On both DNA strands, this occurs at both primer-annealing sites.
The fragment that is being replicated is the target fragment. Repetition of PCR
cycles (in three to four steps about 40 to 50 times) results in the target
amplification between the 5' ends of the two primer binding sites.

A single primer binds to the genomic DNA at two different sites on opposite
strands of the DNA template (Fig.13.6). If these priming sites are within an
amplifiable distance of each other, a discrete DNA product is produced
through amplification. The polymorphisms between individuals result from
sequence differences in one or both of the primer binding sites. The technique
detects nucleotide sequence polymorphisms in a DNA amplification-based
assay using only a single primer of the nucleotide sequence. Amplification
products are separated by gel electrophoresis and visualized by ethidium
bromide staining under gel documentation system.

94 Fig. 13.6: Various steps in the RADP technique.

Unit 13 MolecularTechniques
Welsh and McClelland, 1990 developed a method that known as random
amplified polymorphic DNA (RAPD) (RAPD). It is a PCR-based DNA
fingerprinting technique using primers whose nucleotide sequence is arbitrarily
selected primer at low stringency for first and second strand cDNA synthesis.
Finally, cDNA sequences amplify and electrophoresis are carried out under
different conditions than RAPD.

Single Nucleotide Polymorphisms (SNPs)

SNPs are the genetic markers that represent sites where DNA sequences
differ by a single base. They represent a DNA sequence (a single nucleotide),
abundant in populations, but having a low mutation rate. SNPs give a measure
of the frequency of occurrence of polymorphism at a particular locus, i.e.,
provide an idea about true polymorphism. RFLP or AFLP can detect the
presence of SNPs conducted on PCR products. Methods for SNP detection
require gel-based assays. If SNPs is present at the 3' end of the PCR primer
binding site, it can be detected by the failure of amplification due to a
mismatch between the primer sequence and binding site on the template. The
detection of SNP at the internal position of an amplicon need PCR
amplification followed by a non-gel-based assay (Fig. 13.7). The non-gel-
based SNPs detection assays are based on detecting a mismatch between
the PCR product and oligonucleotides at the internal site as a probe. It helps in
distinguishing the wild and mutant alleles.

Fig. 13.7: Various steps involved in the formation of SNPs. 95

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The polymorphism has been found in the human genome with an estimated
average frequency of one SNP per kilobase pair. Their inheritance's stability
and relative fidelity are higher than the other markers. Therefore, methods for
SNP detection and the construction of the SNP genetic map have been
employed. SNPs have been detected in many crops like barley, wheat, and
maize. Some assays like TaqMan and Molecular beacon used to detect the
SNP in various crops.

a) TaqMan assay- An oligonucleotides probe is labelled with a fluorescent

reporter molecule at the 5’ end and a quencher at the 3' end. The probe
is degraded after hybridization due to the exonuclease activity of the Taq
polymerase enzyme during the extension phase of PCR. A high
fluorescence signal is generated. Due to the SNP, the probe mismatches
the template leading to a failure in duplex formation, no such
degradation at the 5' end of the probe is possible, and no rise in the
fluorescence signal will be detected.

b) Use of Molecular beacon- Molecular beacon is a short oligonucleotide

(25 - 40 nucleotides) that forms a hairpin structure with a loop and stem.
The loop is designed to hybridize specifically to a 15 - 25 nucleotide
portion of the target DNA sequence, while 5 - 6 nucleotide long stem
sequences are complementary to each other. A fluorescent reporter
molecule is attached at the 5’- end of the molecular beacon while a non-
fluorescent quencher is attached at 3’- end. In real-time PCR, during
annealing, binding of molecular beacon to its target leads to increased
fluorescence signal, due to separation of reporter and quencher. If SNP
is available, the probe fails to form a duplex with the template DNA, and
generates a hairpin-like structure due to the self-annealing of its two
ends, thereby quenching the reporter. A sensing device can detect the
very less or no signals fluorescence signal.

b) Oligonucleotide Ligation Assay (OLA) - Two independent probes are

used for hybridization with PCR product. When probes match the
product, the two probes anneal with the PCR product and undergo
ligation resulting in oligonucleotides which is biotinylated at 5’ end, and
labelled at 3’ end. The ligation product is captured on the streptavidin-
coated matrix, and a signal is detected by autoradiography. When SNP
is present, there is a mismatch. The labelled and biotinylated
oligonucleotides are unable to ligate. When captured by streptavidin,
they carry no signal.

c) DNA microarray or chip- An array of oligonucleotides of known

overlapping sequence differing at solitary specific nucleotides can be
used to detect SNPs. Four oligonucleotides that differ at SNP position
are hybridized with biotinylated PCR products. The perfect match will
allow binding while the mismatched products will be washed away.

d) Dynamic allele-specific hybridization – The technique is based on

differences in temperature between duplexes due to perfectly match and
mismatch between PCR product and oligonucleotide. The PCR product
is immobilized on a solid support and denatured to give a single-
96 stranded DNA which is hybridized to an oligonucleotide probe containing
Unit 13 MolecularTechniques
an SNP site. The duplex formed is detected using a fluorescent dye
specific for double-stranded DNA. An increase in temperature denatures
the duplex leading to a reduction in fluorescence. A sudden decrease in
fluorescence indicates a mismatch, thus identifying the SNP.

e) Minisequencing - SNPs can also be detected using the Sanger

dideoxynucleotide method. The oligonucleotide primer has a sequence
of one or more base pairs upstream of the SNP site.

SAQ 2
a) State whether these statements are ‘True ‘or ‘False’.

i) Minisatellites are non-variable sequence elements present in the

genome.

ii) DNA fingerprinting technique helps in the identification of variable

elements within DNA sequences.

iii) RFLP technique depicts the location of different DNA sequences.

iv) RAPD involves using many primers in a PCR reaction resulting in

the amplification of many discrete DNA products.

v) Single Nucleotide Polymorphisms (SNPs) measure the frequency

of occurrence of polymorphism at a particular locus.

b) Define SNPs. Mention their significance.

13.5 DNA SEQUENCING, PCR AND REVERSE

TRANSCRIPTASE-PCR (RT-PCR)
DNA sequencing
Frederick Sanger & Colleagues developed DNA sequencing technique in
1980. First, DNA template molecules obtained from PCR or cloned DNA
fragments are taken. Then, the template DNA is mixed with the primer that is
complementary to the 3' end of one strand of the region to be sequenced. Taq
polymerase, all four deoxyribonucleoside triphosphate precursors (dNTPs),
and a low concentration of modified precursors called dideoxyribonucleoside
triphosphatases (ddNTPs) are all present in the reaction mixture. Each ddNTP
s is modified by adding colored fluorescent dye to its 3’ end.

The reaction begins by heating the mixture to a temperature so that two

strands denature. The reaction is cooled, and the primer can hybridize with the
template DNA. One primer is present so that only one DNA strand can
hybridize with the primer. The Taq polymerase adds dNTPs to the end of the
primer that is complementary to the template molecule, synthesizing a new
complementary strand of DNA. The polymerase inserts a ddNTP instead of
dNTP. Dideoxyribonucleotides lack a hydroxyl group at both 2' and 3'
positions. When the nucleotide gets incorporated into the end of the growing
chain, the lack of 3' OH makes it impossible for the polymerase to add another
nucleotide leading to chain termination. The incorporation of ddNTP ceases
the growth of the chain. 97
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After the extension of the chain is complete, the temperature is raised to
denature the new double-stranded DNA molecules. The cycle of hybridization,
synthesis and denaturation is repeated many times, generating a large
population of daughter DNA strands with ddNTP molecules incorporated at
every position. When the reaction is complete, the products are separated by
gel electrophoresis. High-resolution gel electrophoresis separates fragments
that differ by one nucleotide in length. The bands in the gel contain one
nucleotide longer than those in the previous band. Each ddNTP is labelled
with the fluorescent dye, and the color of the band is read by an automated
laser detector. It reveals the identity of the terminal nucleotide on each
daughter molecule. The order of the colors in the gel corresponds to the base
sequence of the template molecule.

Massive parallel sequencing (2D generation sequencing) has been developed

in the last few years. The fragments are prepared directly from the genome,
and each genome fragment is amplified to form a considerable population.
This technique is based on polymerase dependent DNA synthesis. In this
method, premature termination of strands or separation of strands by
electrophoresis is not required. The nucleotides are identified as they get
incorporated by polymerase in real-time. Automated sequencers use giant
DNA molecules immobilized on the surface and incubated with DNA
polymerase in the presence of dNTPs (Fig.13.8). The nucleotides that get
incorporated in the complementary strands are identified using various
procedures, including DNA sequencing technologies to study individual
genomes from a species. Recently the third-generation sequencers have also
been used. These sequencers act on single DNA molecules (eliminates the
requirement of DNA amplification), generate long reads (about 1000
nucleotides), operate at low cost and a much faster rate. Nucleotide
identification in these sequencers occurs based on ionic properties or
sequencing by the synthesis in which a laser-based detection system identifies
fluorescently labelled nucleotides. Various software systems are used to
analyze the nucleotide sequence of the DNA fragment.

Fig.13.8: Diagrammatic representation of various steps involved in DNA sequencing

98 via Sanger method.
Unit 13 MolecularTechniques
PCR and Reverse Transcriptase-PCR

In 1983, Kary Mullis developed a new technique of amplifying DNA fragments

that is known as Polymerase Chain Reaction (PCR). In this reaction unlimited
copies of a DNA segment can be quickly produced using this technique. DNA
fragments having a single DNA molecule can be amplified many times. This
technique proves beneficial if the DNA sample is small. The technique is
based on the principle that two strands of DNA comprise the double helix
complement each other can be separated at high temperatures and
reannealed at low temperatures, and single-stranded fragments are referred to
as templates. RNA templates can be converted to complementary DNA using
reverse transcriptase.

The Taq polymerase enzyme, four building blocks of DNA- the nucleotides
(ATP, CTP, GTP, TTP), forward and reverse primer sequence, and DNA
template are required for carrying out the PCR (Fig. 13.9). A heat-stable Taq
polymerase isolated from bacterium Thermus aquaticus that lives in hot
springs at a temperature above 90oC. The PCR technique involves repeated
cycles of DNA synthesis or replication. Each cycle comprises three basic
steps- separating the strands, annealing the primer to the template, and
synthesizing new strands. The product of the first cycle becomes the template
for the next cycle.

The oligonucleotides act as primers to which the nucleotides are added during
the replication steps. The mixture is heated to 90oC, which causes the DNA
strands to separate into two strands. The mixture is then cooled to about 60oC.
It allows the primers to hybridize to the strands of the target DNA. The
temperature is then increased to 72oC to allow the polymerase to add
nucleotides at the 3’ end of the primers. The polymerase extends the primer.
The DNA gets copied, forming new complementary strands. The temperature
is raised once more to separate the newly formed complementary strands
from the original DNA strands; the sample is then cooled to allow the synthetic
primers in the mixture to bind to the target DNA once more. The cycle is
repeated repeatedly, each time doubling the specific region of DNA flanked by
the bound primers. Hundreds of copies of a specific region of DNA can be
generated in a few hours using a thermal cycler that automatically changes the
temperature of the reaction mixture to allow each step in the cycle to take
place.

PCR technique can generate many copies of the specific DNA fragment
before cloning the fragment. Therefore, DNA can be amplified without cloning;
hence the procedure is less time-consuming. In about 25 rounds of PCR, more
than one million copies of DNA are produced. If the target sequence is known,
then the nucleotide sequence of the complementary primers can be specified.
PCR technique has been used in criminal investigations to generate the
quantities of DNA from the dried blood sample from the cloth of the suspect or
even a hair follicle. A highly polymorphic genomic region is selected for
amplification because every individual possesses no similar-sized DNA
fragments. PCR can also be used to study the DNA fragments from preserved
fossil remains. The PCR technique finds its application in forensic science,
medical diagnostics, food analysis, and research studies. 99
Block 4 Biotechnology - Techniques and Applications

Fig.13.9: Various steps in the PCR technique.

Reverse Transcriptase-PCR

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is sensitive in

vitro method to amplify RNA sequence. RT-PCR is used to detect and
compare mRNA levels and the surface proteins and a crucial role in medical
science.

Reverse transcription coupled to the polymerase chain reaction (RT-PCR) is

commonly used to detect the presence of mRNAs, pre-mRNAs, or other types
of RNA such as non-coding RNAs. The method involves transcribing a portion
of the RNA genome into complementary DNA (cDNA), then amplified through
PCR. The method involves using a primer annealed to the RNA of interest.
The primer is usually a synthetic oligo(dT)15-18, a random hexamer mixture
(dN)6, or a synthetic DNA oligonucleotide that is complementary to a specific
transcript (a gene-specific primer) (Fig.13.10). The DNA-RNA hybrid serves as
a template during reverse transcription. The reverse enzyme transcriptase
(RT) generates a single-stranded cDNA copy of a portion of the target RNA
molecule. cDNA sequences obtained from mRNAs can be used as a template
for PCR. The use of gene-specific primers produces DNA fragments
100 corresponding to a portion of the RNA of interest.
Unit 13 MolecularTechniques

Fig. 13.10: Various steps in the RT-PCR technique.

SAQ 3
a) Answer in one word.

i) Technique by which unlimited copies of DNA can be produced

ii) Single-stranded DNA fragments.

iii) Heat stable DNA polymerase enzyme.

iv) The technique used for the detection and comparing mRNA levels

v) The enzyme that generates a single-stranded cDNA copy of a part

of the RNA molecule

b) What is the difference between PCR and RT-PCR?

13.6 HYBRIDOMA AND MONOCLONAL

ANTIBODIES
Antibodies are the proteins produced by lymphoid tissues in response to
foreign materials or antigens. Antibodies are specific in their mode of action. 101
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Antibodies bind to selective molecules within the cell, having a small part that
fits into the antigen-antibody binding sites of the antibody. In 1975, Cesar
Milstein and Gorges Kohler carried out experiments related to preparing
antibodies against specific antigens. Antibody producing cells derived from a
single lymphocyte synthesizes antibodies having identical antigen combining
sites. When a single purified antigen is injected into the animal, many different
lymphocytes are activated, and each antibody has an affinity for different parts
of the antigen. If an animal is injected with pure antigen, antibodies are
produced after a week. The spleen or the lymphoid organs are removed from
the animal, and isolates of the single suspension of cells produce the desired
antibodies (Fig.13.11). If these cells are grown as separate colonies, large
quantities of the particular immunoglobulin can be obtained. This procedure
can obtain antibodies produced by a single colony of cells (clone). The
antibodies produced by this procedure are called monoclonal antibodies.

Fig.13.11: Hybridoma technology for production of monoclonal antibodies.

For the production of monoclonal antibodies, the antigen is injected into the
mouse to cause the proliferation of specific antibody-forming cells. After
several weeks, the spleen is removed and dissociated into single cells. The
antibody-forming lymphocytes are fused with a population of malignant
myeloma cells. It makes the hybrid cells immortal, which show capacity for
limited division. Hybrid cells, i.e. hybridomas, are selected from the non-fused
cells by placing the cells in the medium in which only hybrid cells can survive.
Hybridomas are grown clonally in separate wells. They are individually
screened for the production of antibodies against the antigen studied. Hybrid
cells containing the antibody are then cloned in vitro or in vivo to get
monoclonal antibodies.

Malignant myeloma cells are cancer cells that proliferate in culture and
produce large quantities of antibodies. The antibodies are not formed in
response to an antigen in these cells. Instead, Myeloma cells are formed by
102 the random conversion of normal lymphocytes to a malignant state. They form
Unit 13 MolecularTechniques
antibodies that were produced from the lymphocyte before turning into the
myeloma cells. Milstein and Kohler combined the properties of two types of
cells, i.e. normal antibody-producing lymphocyte cells and the immortal
myeloma cell, to produce hybrid calls called a hybridoma. They grow and
proliferate indefinitely and produce large monoclonal (single-cell) antibodies.

Monoclonal antibodies find their use in medical diagnostics to test or

determine the concentration of specific proteins in the blood or urine. For
example, the monoclonal antibodies form the basis of pregnancy confirmation
tests to monitor the presence of a protein–chorionic gonadotropin that appears
in the urine after a few days of conception. Monoclonal antibodies have also
become useful as therapeutic agents in treating various diseases.

13.7 ELISA AND IMMUNODETECTION

Enzyme-linked immunosorbent assay (ELISA) is used to detect specific
antigens or proteins in a biological sample. It is also used to detect viruses,
fungi, bacteria, mycotoxins and hormones. The assay is based on the ability of
low molecular weight antibodies to combine with enzymes to produce
immunological conjugates. The immune reaction is detected with
histochemical staining techniques. The antibody is involved in the immune
reaction, and the conjugated enzyme is used for staining reaction using the
specific substrate. ELISA technique was developed by E. Engvall and P.
Perlmann in 1972. Clarks and Adams (1977) used this technique to detect
viruses by using antibodies. Voller and his colleagues (1980) explored using
a polystyrene or polyvinyl chloride microtitre plate with 96 wells for the assay.
The plate provides a solid phase of the immune reaction. The
antigens/antibodies get stuck on the inner surface of ELISA wells. The
molecular event of the antigen-antibody binding can be visualized by adding
an enzyme to label the antigen or the antibody (Fig. 13.12). The enzymes
such as horseradish peroxidase alkaline phosphatase have been used for this
purpose. The addition of the substrate for the enzyme produces a colored
product that indicates antigen-antibody binding. The optical densities are
measured using an ELISA reader. In DAS (Double Antibody Sandwich) ELISA,
the wells are coated with antibodies or immunoglobulins (Ig). The sample is
added, and time is provided to allow the antigen to bind to the antibody. The
virus antigen gets trapped in the coated antibody. The enzyme labelled
antivirus, i.e., Ig, is added after that. It gets attached to the trapped antigen.
Finally, an enzyme substrate is added to produce the colored product.
Generally, the enzyme peroxidase or alkaline phosphatase is used for the
reaction.

In DAC ELISA, the antigen coating is applied directly to the wells. Diluted
unfractionated antiserum is added. Antibodies or immunoglobulins (Ig) get
attached to the virus antigen can be detected with the help of an enzyme –Ig-
conjugates. In another type of ELISA called PAC-ELISA, the wells are coated
with protein A to which highly diluted unprocessed specific antisera is added.
Test samples and antiserum are added. Finally, the substrate is added to get
the colored reaction. The procedure has been used to detect mycoplasma like
organisms (MLOs) in crude leaf extracts. DAS-LISA has been used for the
quantitative estimation of bacterial populations. ELISA has been used for the
detection of aflatoxins in the seed samples. 103
Block 4 Biotechnology - Techniques and Applications
The ELISA technique is used widely to detect the antigens, antibodies in the
blood, pregnancy, hormone levels, and adulterants in food. In addition, ELISA
diagnostic kits have been created to detect various diseases. It has also been
used to detect mycorrhizal fungi and gibberellins through the use of
monoclonal antibodies.

Fig.13.12: Diagrammatic representation of ELISA technique.

SAQ 4
Define

i) Hybridoma

ii) Monoclonal antibodies

iii) Primer

iv) Probe

13.8 SUMMARY
• DNA, RNA and protein fragments can be separated by gel
electrophoresis. The bands obtained after separation can be hybridized
using a labelled probe. The bands are transferred on the nitrocellulose
membrane. "Blotting" describes the process of transferring bands to a
membrane and then detecting their presence using a probe. The
technique in which DNA bands are blotted is called Southern blotting.
The transfer of RNA bands is called Northern blotting. The transfer of
protein bands is done by the technique called Western blotting. Southern
blot hybridization helps in the detection of specific DNA fragments. In
contrast, Northern blotting helps characterize one or more specific
mRNA transcripts in a sample of total RNA. Western blotting is used for
the detection of proteins of particular specificity.

• DNA fingerprinting technique is used to identify an individual using a

sample of DNA. DNA sequences provide many visual markers that can
be used to map their locations in the chromosome of various species.

• Molecular markers are used to detect changes among different species

and strains. The molecular markers that are extensively used in the
preparation of genome maps include RFLP, RAPD and SNPs. RFLP
markers are based on specific restriction enzymes and molecular
104 probes.
Unit 13 MolecularTechniques
• RAPD markers are DNA fragments from PCR amplification of random
segments of genomic DNA with a single primer of an arbitrary nucleotide
sequence.

• SNPs represent sites where DNA sequences differ by a single base.

They are also referred to as simple nucleotide polymorphisms. RFLP or
AFLP can detect the presence of SNPs conducted on PCR products.
However, number of methods for SNP detection uses gel-based assays.

• Identical template molecules obtained from PCR or cloned DNA

fragments are taken and used as the template along with primer
complementary to the 3' end of one strand of the region to be
sequenced. The nucleotides incorporated in the complementary strands
are identified using various procedures. The strands synthesized by this
procedure are short in length. Therefore, these DNA sequencing
technologies are best suited for studying individual genomes from a
species.

• The amplifying DNA fragments are known as polymerase chain

reactions (PCR). It allows the production of unlimited copies of a DNA
segment quickly. Many copies of the specific DNA fragment can be
created using this technique. Kary Mullis developed the PCR technique
in 1983. Reverse transcriptase PCR (RT-PCR) was developed to amplify
RNA targets. The method involves using a primer annealed to the RNA
of interest.

• The normal antibody-producing lymphocyte cells and the immortal

myeloma cell is fused to produce hybridoma cells. They grow and
proliferate indefinitely and produce large monoclonal (single-cell)
antibodies. First, the antigen is injected into the mouse, where it causes
the proliferation of specific antibody-forming cells. Next, the antibody-
forming lymphocytes are fused with a population of malignant myeloma
cells. Hybrid cells containing the antibody are then cloned in vitro or vivo
to get monoclonal antibodies.

• Enzyme-linked immunosorbent assay (ELISA) technique is used to

detect specific proteins present in a biological sample. It is also used to
detect viruses, fungi, bacteria, mycotoxins and hormones. The low
molecular weight antibodies combine with enzymes to produce
immunological conjugates.

13.9 TERMINAL QUESTIONS

1. Differentiate between Northern and Southern blotting.

2. Explain the technique of Western blotting.

3. Explain the significance of the DNA fingerprinting technique.

4. Enumerate the role of a few molecular markers.

5. Describe the significant steps involved in the amplification of DNA using

the PCR technique.

6. What is the significance of hybridoma technology?

7. How is ELISA technology important for medical diagnostics? 105

Block 4 Biotechnology - Techniques and Applications
13.10 ANSWERS
Self-Assessment Questions
1. a) Southern blotting b) Northern blotting

c) Western blotting d) Molecular probe

2. a) i) False; ii) True; iii) True; iv) False; v) True

b) Refer to Section 13.4

3. a) i) Polymerase chain reaction ii) Templates DNA

iii) Taq Polymerase iv) Reverse transcriptase PCR

v) Reverse transcriptase

b) Refer to Section 13.5.

4. a) Refer to Section 13.6.

b) Refer to Section 13.6.

c) Primer- Primers are single strands of DNA used for initiating the
synthesis of DNA. These sequences are about 20 to 30 bases in
length.

d) Probe- A single-stranded DNA or RNA fragment corresponding to

a gene or DNA sequence of interest is called a probe. It is a
nucleotide sequence that is complementary to the target
sequence.

Terminal Questions
1. Refer to Section 13.2.

2. Refer to Section 13.2.

3. Refer to Section 13.3.

4. Refer to Section 13.4.

5. Refer to Section 13.5.

6. Refer to Section 13.6.

7. Refer to Section 13.7.

Acknowledgement
Fig. 13.8 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fww
w.researchgate.net%2Fprofile%2FMichel-Gauthier-
4%2Fpublication%2F234248746%2Ffigure%2Ffig2%2FAS%3A
669425925623808%401536614986157%2FThe-Sanger-
sequencing-method-in-7-steps-1-The-dsDNA-fragment-is-
denatured-into-two.png&imgrefurl

106