ANIKA JAMES

S6 BC AND IMB
002
 TABLE OF CONTENTS

01 PCR 02 Real Time PCR

Animal Viral
03 04 Plant Viral
05
Vectors Vectors
Polymerase Chain Reaction
 INTRODUCTION
• PCR or Polymerase Chain Reaction is a technique used in molecular
biology and biotechnology to create several copies of a certain DNA or
RNA segment by amplifying the segments.
• This technique was developed in 1983 by Kary Mullis.
• PCR is very simple and inexpensive technique.
 PRINCIPLE OF PCR
• A short segment of DNA is amplified using primer mediated
enzymes.
• DNA Polymerase synthesises new strands of DNA
complementary to the template DNA.
• The DNA polymerase can add a nucleotide to the
pre-existing 3’-OH group only. Therefore, a primer is
required.
• Thus, more nucleotides are added to the 3’ prime end
of the DNA polymerase.
• The target sequence of nucleic acid is denatured to single strands,
primers specific for each target strand sequence are added, and DNA
polymerase catalyzes the addition of deoxynucleotides to extend and
produce new strands complementary to each of the target sequence
strands .
• In the next step, both double-stranded products of the above step are
denatured and subsequently serve as targets for more primer annealing
and extension by DNA polymerase.
• After 25 to 30 cycles, at least 107 copies of target DNA may be
produced by means of this thermal cycling.
 REQUIREMENTS FOR PCR
• DNA Template– The DNA of interest from the sample
• DNA Polymerase–Since the reaction periodically becomes heated to high
temperature, PCR depends upon using a heat-stable DNA polymerase. Many
such heat-stable enzymes from thermophilic bacteria are now available
commercially. The first one and the most commonly used is the Taq
polymerase from the thermophilic bacterium Thermusaquaticus.
• Oligonucleotide Primers-- These are the short stretches of single-stranded DNA
complementary to the 3’ ends of sense and anti-sense strands.
• Deoxyribonucleotide triphosphate– These provide energy for polymerization
and are the building blocks for the synthesis of DNA. These are single units of
bases.
• Buffer System– Magnesium and Potassium provide optimum conditions for
DNA denaturation and renaturation. It is also important for fidelity,
polymerase activity, and stability.
 PCR STEPS
A. (A) EXTRACTION AND DENATURATION OF TARGET NUCLEIC ACID

• For PCR, nucleic acid is first extracted (released) from the

organism or a clinical sample potentially containing the
target organism by heat, chemical, or enzymatic methods.
• Once extracted, target nucleic acid is added to the
reaction mix containing all the necessary components for
PCR (primers, nucleotides, covalent ions, buffer, and
enzyme) and placed into a thermal cycler to undergo
amplification.
 • (B) STEPS IN AMPLIFICATION

• Conventional PCR involves 25 to 50 repetitive cycles, with each cycle

comprising three sequential reactions:

1. DENATURATION
• The reaction mixture is heated to 95°C for a short time period (about 15–
30 sec) to denature the target DNA into single strands that can act as
templates for DNA synthesis.
2. PRIMER ANNEALING

• The mixture is rapidly cooled to a defined temperature which allows the

two primers to bind to the sequences on each of the two strands flanking
the target DNA.
• The two separated strands run in the opposite direction and
consequently there are two primers- a forward primer and a reverse
primer.
• Primers are short, single-stranded sequences of nucleic acid usually 20 to
30 bases long, selected to specifically hybridize (anneal) to a particular
nucleic acid target, essentially functioning like probes.
• Here, the primers bind to their complementary sequences on the
template DNA.
• They serve as the starting point for the synthesis of DNA.
• This annealing temperature is calculated carefully to ensure
that the primers bind only to the desired DNA sequences
(usually around 55oC).
• One primer binds to each strand. The two parental strands do
not re-anneal with each other because the primers are
in large excess over parental DNA.
3. EXTENSION/ ELONGATION
• The temperature of the mixture is raised to 72-80°C (usually) and kept at
this temperature for a pre-set period of time to allow DNA polymerase to
elongate each primer by copying the single-stranded templates.
• Annealing of primers to target sequences provides the necessary
template format that allows the DNA polymerase to add nucleotides to
the 3’ terminus (end) of each primer and extend sequence
complementary to the target template
• Taq polymerase is the enzyme commonly used for primer extension.
• This enzyme is used because of its ability to function efficiently at
elevated temperatures and to withstand the denaturing temperature of
94°C through several cycles.
• The bases are added to the 3’ end of the primer by the Taq polymerase
enzyme.
• This elongates the DNA in the 5’ to 3’ direction.
• It attaches to the primer and adds DNA bases to the single strand. As
a result, a double-stranded DNA molecule is obtained.
• The DNA polymerase adds about 1000bp/minute under optimum
conditions.
• The ability to allow primer annealing and extension to occur at
elevated temperatures without detriment to the polymerase
increases the stringency of the reaction, thus decreasing the chance
for amplification of non-target nucleic acid (i.e., nonspecific
amplification).
• The three steps of the PCR cycle are repeated.
• Thus in the second cycle, the four strands denature, bind primers and are
extended. No other reactants need to be added. The three steps are
repeated for a third cycle and so on for a set of additional cycles.
• By the third cycle, some of the PCR products represent DNA sequence
only between the two primer sites and the sequence does not extend
beyond these sites.
 C. PRODUCT ANALYSIS

• Gel electrophoresis of the amplified product is commonly employed after

amplification.
• The amplified DNA is electrophoretically migrated according to their
molecular size by performing agarose gel electrophoresis.
• The amplified DNA forms clear bands which can be visualized under
• ultra-violet (UV) light.
REAL TIME
PCR
 INTRODUCTION
• A real-time polymerase chain reaction (real-time PCR, or quantitative
polymerase chain reaction (qpcr)) is a laboratory technique of molecular
biology based on the polymerase chain reaction (PCR).
• It is a technique used to monitor the progress of a PCR reaction in real-
time not at its end, as in conventional PCR.
• Real-time PCR can be used quantitatively hence also called quantitative
polymerase chain reaction (qpcr)
• The copies produced after the extension, so-called amplicons, are
re-amplified with the same primers leading thus to exponential
amplification of the DNA molecules.
• After amplification, however, gel electrophoresis is used to analyze the
amplified PCR products and this makes conventional PCR time consuming;
since the reaction must finish before proceeding with the post-PCR
analysis. Real-Time PCR overcomes this problem.
•
 PRINCIPLE OF REAL TIME PCR
• This same principle of amplification of PCR is employed in real-time PCR.
• But instead of looking at bands on a gel at the end of the reaction, the
process is monitored in “real-time”.
• The reaction is placed into a real-time PCR machine that watches the
reaction occur with a camera or detector.
• They all link the amplification of DNA to the generation of fluorescence
which can simply be detected with a camera during each PCR cycle.
• Hence, as the number of gene copies increases during the reaction, so
does the fluorescence, indicating the progress of the reaction.
• Both amplification and product detection are accomplished in one
reaction vessel without ever opening.
• This dramatically reduces the chances of cross-contamination of samples
with the amplified product.
• Compared to conventional PCR assays, real-time PCR instruments are not
only able to measure amplified product (amplicon) as it is made but are
also able to quantitate the amount of product and thereby determine
the number of copies of the target in the original specimen.
• The amount of time required to complete a real-time PCR assay is
significantly less compared to conventional PCR-based assays because
the time needed for the post-PCR detection of amplified product is
eliminated by the use of fluorescent probes.
 qPCR STEPS
The working procedure can be divided into two steps:
(A) AMPLIFICATION
1. Denaturation
• High temperature incubation is used to “melt” double- stranded DNA into
single strands and loosen secondary structure in single-stranded DNA.
• The highest temperature that the DNA polymerase can withstand is
typically used (usually 95°C).
• The denaturation time can be increased if template GC content is high.
2. Annealing
• During annealing, complementary sequences have an opportunity to
hybridize, so an appropriate temperature is used that is based on the
calculated melting temperature ™ of the primers(5°C below the Tm of the
primer).
3. Extension

• At 70-72°C, the activity of the DNA polymerase is optimal, and primer

extension occurs at rates of up to 100 bases per second.
• When an amplicon in real-time PCR is small, this step is often combined
with the annealing step using 60°C as the temperature.
(B)DETECTION METHODS

• The detection is based on fluorescence technology.

• The specimen is first kept in proper well and subjected to thermal cycle
like in the normal PCR.
• The machine, however, in the Real Time PCR is subjected to tungsten or
halogen source that lead to fluoresce the marker added to the sample
and the signal is amplified with the amplification of copy number of
sample DNA.
• The emitted signal is detected by an detector and sent to computer after
conversion into digital signal that is displayed on screen.
• The signal can be detected when it comes up the threshold level (lowest
detection level of the detector).
FLUORESCENCE MARKERS USED IN REAL TIME PCR

A number of real-time PCR methods have been described, but two have
emerged as the most popular.
1. SYBR
2. Taqman probe

SYBR GREEN
• SYBR green is a dye that binds to double-stranded DNA but not to single-
stranded DNA, and, when so bound, fluoresces.
• This is a dye that emits prominent fluorescent signal when it binds at the
minor groove of DNA, nonspecifically.
• Other fluorescent dyes like Ethidium Bromide or Acridine Orange can also
be used but SYBR Green is better used for its higher signal intensity.
• During PCR cycle, as more and more double-stranded product is
generated to which SYBR green dye attach and fluoresces, an increasing
amount of fluorescent signal is generated
• The amount of fluorescence in the reaction at any particular time is
directly related to the number of double-stranded DNA molecules in the
reaction.
• SYBR Green is more preferred than the Taqman Probe as it can provide
information about each cycle of amplification as well as about the
melting temperature which is not obtained from the Taqman probe.
• However, its disadvantage is the lack of specificity as compared to
Taqman Probe.
• that is it will bind and fluoresce all double-stranded products in the
reaction, whether they are specific products, nonspecific products, primer
dimers, or other amplification artefacts.
 Taq-Man PCR
• Taq-Man PCR uses dye-labelled nucleic-acid probe complementary to
an internal segment of the target DNA.
• This dye-labelled probe anneals to one of the template strands close to
and downstream from one of the two PCR primers.
• The probe is labeled with two fluorescent moieties, the reporter
(fluorophore) is attached to the probe’s 5’ end and the quencher
(tetramethylrhodamine (TAMRA)) is attached to its 3’ end.
• When the reporter and the quencher are connected, the quencher
reduces the fluorescent signal of the reporter dye as it absorbs the
energy via a fluorescence resonance energy transfer (FRET).
• However, during PCR, Taq polymerase, extending the primer on the
probe’s target strand, displaces and degrades the annealed probe
through the action of its 5’ to 3’ exonuclease function.
• The fluorophore is thereby released from its molecular attachment to the
quencher and fluoresces.
• As more PCR products are generated, the more dye-labeled probe will
find target regions to join which eventually leads to release of the
reporter molecule during the subsequent amplification process.
• The release of reporter dyes is reflected as increased intensity of the
fluorescent signal which is proportional to the amount of amplicon
synthesized.
• Whether using SYBR green or TaqMan probes, the relationship between
signal intensity and the amount of template in a real-time PCR reaction
provides a reliable means both to quantitate nucleic acids and to assay
for the presence or absence of specific gene sequences.
 VIRAL VECTORS
• Viral vectors are tools commonly used by molecular biologists to deliver
genetic material into cells.
• Viruses have evolved specialized molecular mechanisms to efficiently
transport their genomes inside the cells they infect. Delivery of genes or
other genetic material by a vector is termed transduction and the
infected cells are described as transduced.
• Molecular biologists first harnessed this machinery in the 1970s. Paul
Berg used a modified SV40 virus containing DNA from the
bacteriophage λ to infect monkey kidney cells maintained in culture.
• A vector is a substance, usually a piece of DNA that carries a
sequence of DNA or other genetic material and introduces it into a
new cell.Vectors act as vehicles to transfer genetic material from one
cell to the other for different purposes like multiplying, expressing, or
isolation.
ANIMAL
VIRAL
VECTORS
SV40 as vector:
• SV40 is a well-known virus, vectors are easy-to-make, and can be produced
in titers of 10(12) IU/ml.
• The genome of SV40 contains very little non-essential DNA so it is necessary to
insert the foreign gene in place of essential viral genes and to propagate the
recombinant genome in the presence of a helper virus.
• The early protein coding genes can be replaced by the gene of interest.
Capacity of the transgene is limited to 5 Kb. Long lasting transgene
expression.
• It has been reported that the transgene expression once established will
remain lifelong.
• This virus is capable of infecting several mammalian species, following a lytic
cycle in some host and a lysogenic cycle in others.
• They also efficiently transduce both resting and dividing cells, deliver
persistent transgene expression to a wide range of cell types, and are
nonimmunogenic.
Retroviral vector
• Single stranded RNA genome
• Has two copies of the genome, which resemble eukaryotic mRNAs.
• The viral genome is reverse transcribed by reverse transcriptase into a
DNA double-strand copy inside the host cells. This process is called as
Reverse transcription.
Features of RV vector
• Contains gene for replication, expression and packaging (ψ
sequences).
• Gene of interest may inserted in the nonessential coding region or it may
replace some essential gene (gag).
• genomes are used as vectors, generally as shuttle vectors.
• Retrovirus genomes commonly contain these three open reading frames
that encode for proteins that can be found in the mature virus. Group-
specific antigen (gag) codes for core and structural proteins of the virus,
• polymerase (pol) codes for reverse transcriptase, protease and integrase
and •envelope (env) codes for the retroviral coat proteins.
1.Bovine Papillomavirus DNA Vectors

• Papillomavirus transformed cells don't contain integrated viral DNA rather

they contain between 50 and 300 copies of unintegrated, circular viral DNA.
• The multicopy mode of plasmid replication provides for natural amplification
of cloned genes and, consequently, the possibility of higher levelsof
expression.
• All BPVs have a circular double-stranded DNA genome.
• They can cause skin tumour equine sarcoid in horses and donkeys.
• BPV is capable of transforming certain mammalian cells including rat, bovine,
hamster, and mouse cell lines in culture.
• BPVs are small non-enveloped viruses with an icosahedral capsid around 50–
60 nm in diameter.
PLANT VIRAL
VECTORS
• Exploitation of plant viruses as transformation vectors by massive infection
may be harmful and even deleterious to the target plants
• It is still however able to express and produce foreign proteins.
• Plant viruses must exhibit some of the exemplary features before they are
considered as vectors.
• They should extend their broader host-range, spread of seed transmission
and carry additional copies of gene of interest.
• Several viral vectors require suitable modification in order to
accommodate extra nucleic acid and also aggressive in infection
process.
• Although several groups of viruses have been identified, some moderate
progresses have been made only in two groups.
• These two groups are Caulimo virus and Gemini virus, which have DNA
genome as genetic material.
Cauliflower Mosaic Virus (Caulimovirus):

• Cauliflower mosaic virus (CamV) belongs to the group caulimovirus, can

be used as potential candidate to deliver foreign gene into the plant.
• It is perhaps the best studied viruses among plant virus, which infects
several members belonging to Cruciferae family.
• Cauliflower mosaic virus contains circular double helical DNA as genetic
material.
• As an infective agent, can cause disease in wide range of commercially
important cultivated crops.
• Cauliflower mosaic DNA has been subjected to a wide range of
manipulation.
• This was the only and first virus to be manipulated and used as a
favourable choice for genetic engineering work.
• Elucidation of 8 kb CamV reveals that, it contains six major and two
minor reading frames
Cow Pea Mosaic Virus Expression Vector:

• Cow pea mosaic virus (CpmV) is also a RNA virus and infects species of
legumes.
• There are two separate positive strand-RNA molecules present in the genetic
material of CpmV.
• The number of nucleotides present in the RNA I and RNA II strand is 5889 and
3480, respectively. Although RNA I alone can replicate on its own but both
RNAs are indispensable for infectivity.
• The proteins involved in the replication of the virus are encoded by RNA I
whereas movement proteins are encoded by RNA II. CpmVcapside of both
large (L) and small (S) coat protein of 30 copies each are in isohedral
symmetry. The two capsid proteins are folded into three antiparallel β-barrel
structures.
• In the construction of CpmV expression vector, preference was given to the
replacement of stable chimeras by insertion of foreign sequences rather than
replacement for native residues.
Bacteriophage Lambda Vectors

• Viruses that can infect bacteria

• 1000 times more efficient than plasmid vectors
• Clone DNA fragments in range of 10,000 – 20,000 bps Bacteriophage..
• Soil-based systemic delivery and phyllosphere in vivo propagation of
bacteriophages
• Two possible strategies for improving bacteriophage persistence for
plant disease control
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