PCR Methods and applications

PCR Methods
And Thermostable DNA Polymerases
Behzad Milani
PhD Student of Biochemistry
Supervised by Prof. AmirMozaffari
November 2016

Contents of part I:
• History of PCR
• Polymerase Chain Reaction
• Steps involved
• Applications of PCR
• Factors for optimal PCR
• Variations of PCR methods and their applications
• Comparison PCR & Cloning
• Advantages
• Limitations

History of PCR
• In 1983 Kary Mullis, a scientist working for the
Cetus Corporation was driving along US Route 101 in
northern California when he came up with the idea
for the polymerase chain reaction.
• In 1985 the polymerase chain reaction was
introduced to the scientific community at a
conference in October. Cetus rewarded Kary Mullis
with a $10,000 bonus for his invention.
• Later, during a corporate reorganization, Cetus sold
the patent for the PCR process to a pharmaceutical
company Hoffmann-LaRoche for $300 million.
• In 1993 Mullis awarded nobel prize in Chemistry
along with Michael Smith for his work on PCR.

Polymerase Chain Reaction
• PCR targets and amplifies a specific region of a
DNA strand.
• It is an invitro technique to generate large
quantities of a specified DNA.
• Often, only a small amount of DNA is available
eg.A drop of blood, Semen strains, Single hair,
vaginal swabs etc.
• Two methods currently exist for amplifying the
DNA or making copies
 Cloning—takes a long time for enough clones to
reach maturity
 PCR—works on even a single molecule quickly

Requirements of PCR
• DNA Template
• Primers
• Taq polymerase
• Deoxynucleoside
• Triphosphates(dNTPs)
• Buffer solution
• Divalent cations(eg.Mg2+ )

Steps Involved
Denaturation
• The reaction mixture is heated to a
temperature between 90-98°C so that the
ds DNA is denatured into single strands by
disrupting the hydrogen bonds between
complementary bases.
• Duration of this step is 1-2 mins.

Steps Involved
Annealing
• Temperature of reaction mixture is cooled
to 45-60°C
• Primers are jiggling around caused
by ???????
• Primers base pair with the complementary
sequence in the DNA.
• Hydrogen bonds reform.
• Annealing fancy word for renaturing.

Steps Involved
Extension
• The temperature is now shifted to 72°C
which is ideal for polymerase.
• Primers are extended by joining the bases
complementary to DNA strands.
• Elongation step continues where the
polymerase adds dNTP's from 5' to 3',
reading the template from 3' to 5' side,
bases are added complementary to the
template.
• Now first cycle is over and next cycle is
continued ,as PCR machine is automated
thermocycler the same cycle is repeated up
to 30-40 times.

Optimal PCR Factors
 PCR Primers
 DNA Polymerase
 Annealing Temperature
 Melting Temperature
 G/C content

Optimal PCR Factors
 PCR Primers
 correctly designed pair of primers is required
 primer dimer,hairpin formation should be
prevented
 length of primer
 DNA Polymerase
 G/C content

Optimal PCR Factors
 PCR Primers
 DNA Polymerase
 Thermus aquaticus (Taq - 170°F)
 Taq polymerase is heat resistant
 It lacks proof reading exonuclease activity
 Other polymerases can be used, eg:
 Tma DNA Polymerase from Thermotoga
maritama,
 Pfu DNA Polymerase from Pyrococcus furiosus.
 G/C content

Optimal PCR Factors
 PCR Primers
 DNA Polymerase
 Very important since the success and specificity
of PCR depend on it because DNA-DNA
hybridization is a temperature dependent process.
 If annealing temperature is too high, pairing
between primer and template DNA will not take
place then PCR will fail.
 Ideal Annealing temperature must be low enough
to enable hybridization between primer and
template but high enough to prevent
amplification of nontarget sites.
 Should be usually 1-2°C or 5°C lower than
melting temperature of the template-primer
duplex
 G/C content

Optimal PCR Factors
 PCR Primers
 DNA Polymerase
 Temperature at which 2 strands of the duplex
dissociate.
 It can be determined experimentally or calculated
from formula
Tm = (4(G+C)) + (2(A+T))
 G/C content

Optimal PCR Factors
 PCR Primers
 DNA Polymerase
 G/C content
 Ideally a primer should have a near random mix
of nucleotides, a 50% GC content
 There should be no PolyG or PolyC stretches that
can promote non-specific annealing

Applications of PCR
 Molecular Identification
 Sequencing
 Genetic Engineering

Applications of PCR
 Molecular Archaeology
 Molecular Epidemiology
 Molecular Ecology
 DNA fingerprinting
 Classification of organisms
 Genotyping
 Pre-natal diagnosis
 Mutation screening
 Drug discovery
 Genetic matching
 Detection of pathogens
 Sequencing

Applications of PCR
 Sequencing
 Bioinformatics
 Genomic cloning
 Human Genome Project

Applications of PCR
 Sequencing
 Site-directed mutagenesis
 Gene expression studies

Variations of PCR
PCR is highly versatile technique and has been
modified in variety of way to suit specific
applications.

Variations of PCR
 Inverse PCR
 In this method amplification of DNA of unknown
sequence is carried out from known sequence.
 This is especially useful in identifying flanking
sequences of various genomic inserts.
 The inverse PCR method includes a series of
digestions and self-ligations with the DNA being
cut by a restriction endonuclease. This cut results
in a known sequence at either end of unknown
sequences.
 Inverse PCR uses standard PCR however it has
the primers oriented in the reverse direction of
the usual orientation.
 The template for the reverse primers is a
restriction fragment that has been ligated upon
itself to form a circle.

Variations of PCR
 Inverse PCR
Inverse PCR Steps:
 Target DNA is lightly cut into smaller fragments of
several kilobases by restriction endonuclease
digestion.
 Self-ligation is induced under low concentrations
causing the phosphate backbone to reform. This
gives a circular DNA ligation product.
 Target DNA is then restriction digested with a
known endonuclease. This generates a cut within
the known internal sequence generating a linear
product with known terminal sequences. This can
now be used for PCR.
 Standard PCR is conducted with primers
complementary to the now known internal
sequences.

Variations of PCR
 Inverse PCR

Variations of PCR
 Ligation-Mediated PCR (LM-PCR)
 Ligation-mediated PCR uses small DNA
oligonucleotide 'linkers' (or adaptors) that
are first ligated to fragments of the target DNA.
 PCR primers that anneal to the linker sequences
are then used to amplify the target
fragments.
 This method is deployed for DNA sequencing,
genome walking, and DNA foot-printing.
 The principle of Ligation Mediated PCR (LM-PCR).
1-Ligation with excess of primers,
2-Polymerase chain reaction of individual
fragments.

Variations of PCR
 In LM-PCR, each fragment is amplified
independently so that due to intrinsic
differences among individual fragments, some
fragments are amplified less efficiently than
others. This results in non-uniform
representation of original genetic material in the
resultant amplicon, which consequently leads to
loss of genetic information and inaccurate
results.
 Primer-extension step (Step 3): a gene-specific
primer (Primer 1) was annealed at 48°C and the
primer was extended with Sequenase enzyme at
48°C.
 Ligation step (Step 4): all extended DNA
fragments with a blunt-end and 5'-phosphate
group were ligated to an unphosphorylated
synthetic asymmetric double-strand linker.

Variations of PCR
 Linear amplification step (Step 5): a second
gene-specific primer (Primer 2) was annealed to
DNA fragments for a one-cycle extension using
Taq DNA polymerase.
 Exponential amplification step (Step 6): the
primer 2 and the linker primer (the longest of the
two oligonucleotides of the linker) were used to
exponentially and specifically amplify DNA
fragments.
 Sequencing gel electrophoresis and
electroblotting (Step 7): amplified DNA fragments
were size-separated on a denaturing 8%
polyacrylamide gel and transferred onto a nylon
membrane by electroblotting.
 Hybridization (Step 8): the nylon membrane was
hybridized overnight with a gene-specific probe.

Variations of PCR
Uses:
 Is the most sensitive sequencing technique
available to map single-stranded DNA breaks at
the nucleotide level of resolution using genomic
DNA.
 LM-PCR has been adapted to map DNA damage
and reveal DNA–protein interactions inside living
cells.
 However, the sequence context (GC content), the
global break frequency and the current
combination of DNA polymerases used in LM-PCR
affect the quality of the results.

Variations of PCR

Variations of PCR
 Multiplex Ligation-dependent Probe
Amplification PCR (MLPA-PCR)
 MLPA is used to establish the copy number of up
to 45 nucleic acid sequences in one single
multiplex reaction. The method can be used for
genomic DNA (including both copy number
detection and methylation quantification) as well
as for mRNA profiling, it permits multiple targets
to be amplified with only a single primer pair,
thus avoiding the resolution limitations of
multiplex PCR.
 The principle of MLPA is based on the
identification of target sequences by hybridization
of pairs of MLPA probes that bind to adjacent
sequences and can then be joined by a ligation
reaction. In order to make one copy of each
target sequence, specific MLPA probes are added
to a nucleic acid sample for each of the
sequences of interest.

Variations of PCR
 The sequences are then simultaneously amplified
with the use of only one primer pair, resulting in a
mixture of amplification products, in which each
PCR product of each MLPA probe has a unique
length.
 One PCR primer is fluorescently or isotopically
labelled so that the MLPA reaction products can
be visualized when electrophoresed on a capillary
sequencer or a gel. Resulting chromatograms
show size-separated fragments ranging from 130
to 490 bp.
 The peak area or peak height of each
amplification product reflects the relative copy
number of that target sequence.
 Comparison of the electrophoresis profile of the
tested sample to that obtained with a control
sample enables the detection of deletions or
duplications of genomic regions of interest

Variations of PCR

Variations of PCR
 Multiplex PCR
 Multiplex PCR is a widespread molecular biology
technique for amplification of multiple targets in a
single PCR experiment.
 In a multiplexing assay, more than one target
sequence can be amplified by using multiple
primer pairs in a reaction mixture.
 As an extension to the practical use of PCR, this
technique has the potential to produce
considerable savings in time and effort within the
laboratory without compromising on the utility of
the experiment.
 Annealing temperatures for each of the primer
sets must be optimized to work correctly within a
single reaction, and amplicon sizes, i.e., their
base pair length, should be different enough to
form distinct bands when visualized by gel
electrophoresis.

Variations of PCR
 Multiplex PCR
Types of Multiplex PCR:
1. Single template PCR reaction; this
technique uses a single template which can be a
genomic DNA along with several pairs of forward
and reverse primers to amplify specific regions
within a template
2. Multiple template PCR reaction; this
technique uses multiple templates and several
primer sets in the same reaction tube. Presence
of multiple primer may lead to cross
hybridization with each other and the possibility
of mis-priming with other templates.

Variations of PCR
 Multiplex PCR
Primer Design Parameters for Multiplex PCR:
Design of specific primer sets is essential for a successful
multiplex reaction. The important primer design
considerations described below are a key to specific
amplification with high yield.
• Primer Length: Multiplex PCR assays involve designing of
large number of primers, hence it is required that the
designed primer should be of appropriate length. Usually,
primers of short length, in the range of 18-22 bases are
used.
• Melting Temperature: Primers with similar Tm, preferably
between 55°C-60°C are used. For sequences with high GC
content, primers with a higher Tm (preferably 75°C-80°C)
are recommended. A Tm variation of between 3°-5° C is
acceptable for primers used in a pool.
• Specificity: It is important to consider the specificity of
designed primers to the target sequences, while preparing a
multiplex assay, especially since competition exists when
multiple target sequences are in a single reaction vessel.
• Avoid Primer Dimer Formation: The designed primers
should be checked for formation of primer dimers, with all
the primers present in the reaction mixture. Dimerization
leads to unspecific amplification.

Variations of PCR
 Multiplex PCR

Variations of PCR
 Multiplex PCR
Advantages of Multiplex PCR:
1. Internal Controls: Potential problems in a simple PCR include
false negatives due to reaction failure or false positives due to
contamination. False negatives are often revealed in multiplex
assays because each amplicon provides an internal control for
the other amplified fragments.
2. Efficiency: The expense of reagents and preparation time is
less in multiplex PCR than in systems where several tubes of
muniplex PCRs are used. A multiplex reaction is ideal for
conserving costly polymerase and templates in short supply.
3. Indication of Template Quality: The quality of the template
may be determined more effectively in multiplex than in a
simple PCR reaction.
4. Indication of Template Quantity: The exponential amplification
and internal standards of multiplex PCR can be used to assess
the amount of a particular template in a sample. To quantitate
templates accurately by multiplex PCR, the amount of
reference template, the number of reaction cycles, and the
minimum inhibition of the theoretical doubling of product for
each cycle must be accounted.

Variations of PCR
 Multiplex PCR
Uses of Multiplex PCR:
Its has been found useful in:
 Pathogen Identification,
 High Throughput SNP Genotyping,
 Mutation Analysis,
 Gene Deletion Analysis,
 Template Quantification,
 Linkage Analysis,
 RNA Detection,
 Forensic Studies.

Variations of PCR
 Methylation-Specific PCR (MSP)
• Methylation-specific PCR (MSP) is used to identify
patterns of DNA methylation at cytosine-guanine
(CpG) islands in genomic DNA .
• Target DNA is first treated with sodium bisulphite,
which converts unmethylated cytosine bases to
uracil, which is complementary to adenosine in
PCR primers.
• Two amplifications are then carried out on the
bisulphite-treated DNA: One primer set anneals
to DNA with cytosines (corresponding to
methylated cytosine), and the other set anneals
to DNA with uracil (corresponding to
unmethylated cytosine).
• MSP used in Q-PCR provides quantitative
information about the methylation state of a
given CpG island.

Variations of PCR
• Treatment of DNA with bisulphite
converts cytosine residues to uracil,
but leaves 5-methylcytosine
residues unaffected.
• Thus, bisulphite treatment
introduces specific changes in the
DNA sequence that depend on the
methylation status of individual
cytosine residues, yielding single-
nucleotide resolution information
about the methylation status of a
segment of DNA.
• The objective of this analysis is
therefore reduced to differentiating
between single nucleotide
polymorphisms (cytosines and
thymidine) resulting from
bisulphite conversion.

Variations of PCR
• The MethyLight method is based on MSP, but
provides a quantitative analysis using real-time
PCR.
• Methylated-specific primers are used, and a
methylated-specific fluorescence reporter probe is
also used that anneals to the amplified region.
• In alternative fashion, the primers or probe can
be designed without methylation specificity if
discrimination is needed between the CpG pairs
within the involved sequences.
• Quantitation is made in reference to a methylated
reference DNA. A modification to this protocol to
increase the specificity of the PCR for successfully
bisulphite-converted DNA (ConLight-MSP) uses
an additional probe to bisulphite-unconverted
DNA to quantify this non-specific amplification.

Variations of PCR

Variations of PCR
 Hot Start PCR
 This is a technique that reduces non-specific
amplification during the initial set up stages of
the PCR
 The technique may be performed manually by
heating the reaction components to the melting
temperature (e.g., 95°C) before adding the
polymerase
 Specialized enzyme systems have been
developed that inhibit the polymerase's activity at
ambient temperature, either by the binding of
an antibody or by the presence of covalently
bound inhibitors that only dissociate after a high-
temperature activation step
 DNA Polymerase- Eubacterial type I DNA
polymerase, Pfu
 These thermophilic DNA polymerases show a very
small polymerase activity at room temperature.

Variations of PCR
 Nested PCR
 This PCR increases the specificity of DNA
amplification, by reducing background due to
non-specific amplification of DNA.
 Two sets (instead of one pair) of primers are
used in two successive PCRs.
 In the first reaction, on pair of primers “outer pair”
is used to generate DNA products, which besides
the intended target, may still consist of non-
specifically amplified DNA fragments.
 The product(s) are then used in a second PCR
after the reaction is diluted with a set of second
set “nested or internal” primers whose binding
sites are completely or partially different from
and located 3' of each of the primers used in the
first reaction.
 The specificity of PCR is determined by the
specificity of the PCR primers.

Variations of PCR
 Nested PCR
 For example, if your primers bind to more than
one locus (e.g. paralog or common domain), then
more than one segment of DNA will be amplified.
To control for these possibilities, investigators
often employ nested primers to ensure specificity.
 Nested PCR means that two pairs of PCR primers
were used for a single locus.
 The first pair amplified the locus as seen in any
PCR experiment.
 The second pair of primers (nested primers) bind
within the first PCR product and produce a second
PCR product that will be shorter than the first one.
 The logic behind this strategy is that if the wrong
locus were amplified by mistake, the probability
is very low that it would also be amplified a
second time by a second pair of primers.

Variations of PCR
 Nested PCR

Variations of PCR
 Nested PCR
Nested PCR strategy:
 Segment of DNA with dots representing non-
discript DNA sequence of unspecified length. The
double lines represent a large distance between
the portion of DNA illustrated in this figure. The
portions of DNA shown with four bases in a row
represent PCR primer binding sites, though real
primers would be longer.
 The first pair of PCR primers (blue with arrows)
bind to the outer pair of primer binding sites and
amplify all the DNA in between these two sites

Variations of PCR
 Nested PCR
 PCR product after the first round of amplificaiton.
Notice that the bases outside the PCR primer pair
are not present in the product.
 PCR product after the first round of amplificaiton.
Notice that the bases outside the PCR primer pair
are not present in the product.

Variations of PCR
 Nested PCR
 Final PCR product after second round of PCR. The
length of the product is defined by the location of
the internal primer binding sites.

Variations of PCR
 Nested PCR
Uses of Nested PCR:
 When a complete genome sequence is known, it
is easier to be sure you will not amplify the wrong
locus but since very few of the world's genomes
have been sequenced completely, nested primers
will continue to be an important control for many
experiments.

Variations of PCR
 AFLP PCR
 AFLP is a highly sensitive PCR-based method for
detecting polymorphisms in DNA. AFLP can be
also used for genotyping individuals for a large
number of loci
Genomic DNA is digested with one or more restriction
enzymes. tetracutter (MseI) and a hexacutter (EcoRI)
Ligation of linkers to all restriction fragments
Pre-selective PCR is performed using primers which
match the linkers and restriction site specific
sequences
Electrophoretic separation and amplicons on a gel
matrix, followed by visualisation of the band pattern

Variations of PCR
 AFLP PCR

Variations of PCR
 Anchored PCR
 A small sequence of nucleotides can be attached
or tagged to target DNA.
 The anchor is frequently a poly G to which a poly
C primer is used.

Variations of PCR
 Anchored PCR

Variations of PCR
 Revers Transcription PCR (RT-PCR)
 A PCR designed for amplifying DNA from RNA.
 Reverse transcriptase reverse transcribes RNA
into cDNA, which is then amplified by PCR.
 RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify
the sequence of an RNA transcript, including
transcription start and termination sites.
 If the genomic DNA sequence of a gene is known,
RT-PCR can be used to map the location of exons
and introns in the gene.
 The 5' end of a gene (corresponding to the
transcription start site) is typically identified by
RACE-PCR (Rapid Amplification of cDNA Ends)

Variations of PCR
 Revers Transcription PCR (RT-PCR)

Variations of PCR
 RACE PCR
 Used to obtain 3' and 5' end sequence of cDNA
transcripts.

Variations of PCR
 RACE PCR

Variations of PCR
 Quantitative Real Time PCR (QRT-PCR)
It is used to amplify and also for quantification
and detection of DNA sample.
 Real time PCR using DNA dyes, such as
Sybr Green, EvaGreen
 Fluorescent reporter probe method, such
as TaqMan,
• Detection and quantitation of fluorescent reporter
the signal of which increases in direct proportion
to the amount of PCR product in a reaction
• Does not measure the amount of end product but
its production in real time

Variations of PCR

Variations of PCR
TagMan Probes PCR:
 TaqMan probes are designed such that they
anneal within a DNA region amplified by a specific
set of primers.
 As the Taq polymerase extends the primer and
synthesizes the nascent strand, the 5' to
3‘ exonulease activity of the polymerase degrades
the probe that has annealed to the template.
 Degradation of the probe releases the fluorophore
from it and breaks the close proximity to the
quencher, thus relieving the quenching effect and
allowing fluorescence of the fluorophore.
 Fluorescence detected in the real-time PCR
thermal is directly proportional to the fluorophore
released and the amount of DNA template
present in the PCR.

Variations of PCR
TagMan Probes PCR:

Variations of PCR
 Asymmetric PCR
 This reaction preferentially amplifies one DNA
strand in a double-stranded DNA template.
 It is used in sequencing and hybridization
probing where amplification of only one of the
two complementary strands is required.
 PCR is carried out as usual, but with a great
excess of the primer for the strand targeted for
amplification.
 Because of the slow (arithmetic) amplification
later in the reaction after the limiting primer has
been used up, extra cycles of PCR are required.
 A recent modification on this process, known as
Linear-After-The-Exponential-PCR (LATE-PCR),
uses a limiting primer with a higher melting
temperature (Tm) than the excess primer to
maintain reaction efficiency as the limiting primer
concentration decreases mid-reaction .

Variations of PCR
 Thermal Asymmetric Interlaced PCR (TAIL-PCR)
This reaction is applied in the isolation of an
unknown sequence flanking a known sequence.
Within the known sequence, TAIL-PCR uses a nested
pair of primers with differing annealing
temperatures; a degenerate primer is used to
amplify in the other direction from the unknown
sequence .
Uses: TAIL-PCR as a powerful tool for amplifying
insert end segments from P1, BAC and YAC
clones, the amplified products were highly specific
and suitable as probes for library screening and
as templates for direct sequencing while the recover
insert ends can also be used for chromosome
walking and mapping.

Variations of PCR
• Nested, insertion-specific primers are used together
with arbitrary degenerate primers (AD primers),
which are designed to differ in their annealing
temperatures.
• Alternating cycles of high and low annealing
temperature yield specific products bordered by an
insertion-specific primer on one side and an
AD primer on the other.
• Further specificity is obtained through subsequent
rounds of TAIL-PCR, using nested insertion-specific
primers.
• The increasing availability of whole genome
sequences renders TAIL-PCR an attractive tool to
easily identify insertion sites in large genome
tagging populations through the direct sequencing
of TAIL-PCR products.
• For large-scale functional genomics approaches, it is
desirable to obtain flanking sequences for each
individual in the population in a fast and cost-
effective manner.

Variations of PCR

Variations of PCR
 Assembly PCR or Polymerase Cycling Assembly
(PCA)
 This entails the artificial synthesis of long DNA
sequences by performing PCR on a pool of long
oligonucleotides with short overlapping segments.
 The oligonucleotides alternate between sense and
antisense directions, and the overlapping
segments determine the order of the PCR
fragments, thereby selectively producing the final
long DNA product.

Variations of PCR
 Assembly PCR or Polymerase Cycling Assembly
(PCA)

Variations of PCR
 In-Situ PCR (ISH)
 A polymerase chain reaction that actually takes
place inside the cell on a slide. In situ PCR
amplification can be performed on fixed tissue or
cells.
Uses:
 Detection and diagnosis of viruses and other
infectious agents in specific cell types within
tissues.
 Detection and characterization of tumor cells
within tissue.
 Detection and diagnosis of genetic mutations in
inherited diseases.
 Detection of gene and gene expression in a tissue.
 Any assay in which identification of cells
expressing a target gene is required. Main
advantages are low background, high specificity,
fast assay with shorter turn-around time and no
need of radioactive chemicals.

Variations of PCR
 In-Situ PCR (ISH)

Variations of PCR
 Allel-Specific PCR
 Selective PCR amplification of the alleles to detect
single nucleotide polymorphism (SNP)
 Selective amplification is usually achieved by
designing a primer such that the primer will
match or mismatch one of the alleles at the 3’
end of the primer.

Variations of PCR
 Allel-Specific PCR

Variations of PCR
 Single Cell PCR
 It is now possible to amplify and examine minute
quantities of rare genetic material, the limit of
this exploration being the single cell.
 Single cell PCR has applications in many areas,
and has great application especially in the field of
prenatal diagnostics.
 In prenatal diagnosis, single cell PCR has made
possible preimplantation genetic analysis and the
use of fetal cells enriched from the blood of
pregnant women for the assessment of single-
gene Mendelian disorders.
 Single-cell PCR has not only proven its usefulness
in diagnostics, but also lately has been very
useful to basic scientists investigating
immunological, neurological and developmental
problems.

Variations of PCR
 Helicase-Dependent Amplification
 This PCR is similar to traditional PCR, but uses a
constant temperature rather than cycling through
denaturation and annealing/extension cycles.
 DNA helicase, an enzyme that unwinds DNA, is
used in place of thermal denaturation.
 Alu PCR
 The pcr is performed using Alu primers designed
to have recognition sequence of Alu restriction
enzyme. Used as a method of obtaining a
fingerprint of bands from an uncharacterized
human DNA.

Variations of PCR
 LONG PCR
 Long PCR is a PCR is which extended or longer
than standard PCR, meaning over 5 kilobases
(frequently over 10 kb). Long PCR is usually only
useful if it is accurate. Thus, special mixtures of
proficient polymerases along with accurate
polymerases such as Pfu are often mixed together.
Applications of Long PCR:
 Long PCR is often used to clone larger genes or
large segments of DNA which standard PCR
cannot.

Variations of PCR
 Arbitrarily Primed PCR (AP-PCR)
Arbitrarily Primed PCR (AP-PCR) or Random
Amplified Polymorphic DNA (RAPD) are
methods of creating genomic fingerprints from
species of which little is known about target
sequence to be amplified.
 TAP-PCR
AP-PCR run at three different annealing
temperature

Variations of PCR
 Colony PCR
 The screening of bacterial (E.Coli) or yeast clones
for correct ligation or plasmid products.
 Selected colonies of bacteria or yeast are picked
with a sterile toothpick or pipette tip from a
growth (agarose) plate.
 This is then inserted into the PCR master mix or
pre-inserted into autoclaved water.
 PCR is then conducted to determine if the colony
contains the DNA fragment or plasmid of interest.

Variations of PCR
 LAMP (Loop-Mediated isothermal amplification)
Assay:
 It is a Modified type of the PCR using 3-6 primers
sets one of them is loop like primer.
 This test use Bst-polymerase (Bacillus
stearothermophilus DNA Polymerase) enzyme.
 Using only two temperatures (63°C and 85°C for
one hour), may be carry out in water bath.

Variations of PCR
 The Digital PCR
 The Digital polymerase chain reaction simultaneously
amplifies thousands of samples, each in a separate
droplet within an emulsion .
 Overlap-Extention PCR
 A genetic engineering technique allowing the
construction of a DNA sequence with an alteration
inserted beyond the limit of the longest practical
primer length .
 Solid Phase PCR
 Encompasses multiple meanings, including Colony
Amplification (where PCR colonies are derived in a gel
matrix, for example), 'Bridge PCR' (primers are
covalently linked to a solid-support surface),
conventional Solid Phase PCR (where Asymmetric PCR
is applied in the presence of solid support bearing
primer with sequence matching one of the aqueous
primers) and Enhanced Solid Phase PCR (where
conventional Solid Phase PCR can be improved by
employing high Tm and nested solid support primer
with optional application of a thermal 'step' to favour
solid support priming)

Variations of PCR
 Box PCR
 Box elements are repetitive sequence elements in
bacterial genome such as Streptococcus genome.
Single primer targeting to the repeats can be used to
fingerprint bacterial species.
 Competitive PCR(cPCR)
 This is a method used for quantifying DNA using real
time PCR. A competitor internal standard is co
amplified with the target DNA and the target is
quantified from the melting curves of the target and its
competitor.
 Consensus PCR
 This PCR is carried out by using flanking primers to
amplify repeat regions from a no. of species. In this
case degenerate/consensus primers can be used for
amplifying the flanking sequences.

Variations of PCR
 Degenerate PCR
 In this instead of using specific PCR primers for a given
sequence, mixed PCR primers will be used.
 That is “wobbles” are inserted into the primers in case
if the exact sequence of gene is not known, so that
there will be more than one possibility for exact
amplifications.
 Degenerate PCR has proven to be a powerful tool to
find ‘new’ gene or gene families. By aligning the
sequences from a no. of related proteins the conserved
and variables part can be determined.
 Based on this information one can use conserved
protein motifs for starting points for designing
degenerate PCR primers.
 Degenerate oligonucleotide-primed PCR(DOR PCR)
 PCR amplification of limited sample by using
degenerate PCR primers is called DOR PCR.

Variations of PCR
 Differential Display PCR (DD PCR)
 It is used for cloning purpose; it combines the
comparative analysis of several samples with the
sensitivity of PCR.
 Forensic PCR
 The VNTR locus is PCR amplified to compare DNA
samples from different sources.
 Hairpin PCR
 A method for error free DNA amplification for mutation
detection. It first converts a DNA sequence to a hairpin.
True mutations will maintain hairpin structure during
amplification while PCR errors will disrupt the hairpin
structure.
 PCR ELISA
 The PCR products are labeled(digoxigenin) during
amplification. A capture probe specific to PCR amplicon
is used to immobilize the amplicon to immune-well
plate. ELISA is then used against the label(anti-
digoxigenin) to quantitate PCR products.

Variations of PCR
 Touchdown PCR (Step-Down PCR)
 A variant of PCR that aims to reduce nonspecific
background by gradually lowering the annealing
temperature as PCR cycling progresses. The annealing
temperature at the initial cycles is usually a few
degrees (3-5°C) above the Tm of the primers used,
while at the later cycles, it is a few degrees (3-5°C)
below the primer Tm. The higher temperatures give
greater specificity for primer binding, and the lower
temperatures permit more efficient amplification from
the specific products formed during the initial cycles.
 Miniprimer PCR
 This reaction uses a thermostable polymerase (S-Tbr)
that can extend from short primers ("smalligos") as
short as 9 or 10 nucleotides. This method permits PCR
targeting to smaller primer binding regions, and is
used to amplify conserved DNA sequences, such as the
16S (or eukaryotic 18S) rRNA gene.

Variations of PCR
 Rep-PCR
 Is used for Genomic Fingerprinting of plant-
associated bacteria and computer-assisted plant
analyses. The genomic fingerprinting method
employed is based on the use of DNA primers
corresponding to naturally occurring interspersed
repetitive elements in bacteria such as REP,ERIC and
BOX elements.
 Vectorette-PCR
 This method enables the amplification of specific
DNA fragments in situation where sequence of only
one primer is known. Thus it extends the application
of PCR to the stretches of DNA where the sequence
information is only available at one end.

Variations of PCR
 Universal Fast Walking PCR
Used for genome walking and genetic fingerprinting using
a more specific 'two-sided' PCR than conventional 'one-
sided' approaches (using only one gene-specific primer
and one general primer - which can lead to artefactual
'noise') by virtue of a mechanism involving lariat structure
formation. Streamlined derivatives of UFW are LaNe RAGE
(lariat-dependent nested PCR for rapid amplification of
genomic DNA ends), 5'RACE LaNe and 3'RACE LaNe .
 Variable Number of Tandem Repeats (VNTR) PCR
This method targets areas of the genome that exhibit
length variation. The analysis of the genotypes of the
sample usually involves sizing of the amplification
products by gel electrophoresis. Analysis of smaller VNTR
segments known as Short Tandem Repeats (or STRs) is
the basis for DNA Fingerprinting databases such as
CODIS .

Variations of PCR
 Intersequence-Specific PCR (ISSR-PCR)
This is a method for DNA fingerprinting that uses primers
selected from segments repeated throughout a genome to
produce a unique fingerprint of amplified product lengths.
The use of primers from a commonly repeated segment is
called Alu-PCR, and can help amplify sequences adjacent
(or between) these repeats.

Variations of PCR
 Other types of PCR
 Overlap extension PCR
 Solid phase PCR
and so on…………………..

Comparison PCR & Cloning
Parameter PCR Gene cloning
1. Final result Selective amplification
of specific sequence
Selective amplification
of specific sequence
2. Manipulation In vitro In vitro and in vivo
3. Selectivity of the
specific segment
from complex
DNA
First step Last step
4. Quantity of
starting material
Nanogram (ng) Microgram (m)
5. Biological
reagents required
DNA polymerase
(Taq polymerase)
Restriction enzymes,
Ligase, vector. bacteria
6. Automation Yes No
7. Labour intensive No Yes
8. Error probability Less More
9. Applications More Less
10. Cost Less More
11. User’s skill Not required Required
12. Time for a typical
experiment
Four hours Two to four days

Advantages of PCR
 PCR in clinical diagnosis
 PCR in DNA sequencing
 PCR in Forsenic Medicine
 PCR in Gene manipulation and expression
studies
 PCR in comparative study of genomics
 PCR in comparison with gene cloning

Limitations of PCR
 Sequence Information
 Amplicon size
 Error rate during amplification
 Sensitivity to inhibitors
 Contamination
 Artefacts

Part II:
Thermostable DNA Polymerases

Contents of part II:
• Discovery
• Properties
• Taq DNA Polymerase
• Other Thermostable Polymerases
• The Error Rate
• Reliability / Fidelity

Discovery
• The original report of this
enzyme, purified from the hot
springs bacterium Thermus
aquaticus, was published in
1976.
• Roughly 10 years later, the
polymerase chain reaction was
developed and shortly
thereafter "Taq" became a
household word in molecular
biology circles.
• *THE DARNDEST PLACES: Scientists
isolated the thermostable DNA polymerase
Taq, an enzyme that drives PCR, from
Thermus aquaticus Yellowstone type-1, a
resident of geysers like this one at
Yellowstone National Park.

Properties
• The thermophilic DNA polymerases, like other DNA
polymerases, catalyze template-directed synthesis
of DNA from nucleotide triphosphates.
• A primer having a free 3‘ hydroxyl is required to
initiate synthesis
• Magnesium ion is necessary.
• In general, they have maximal catalytic activity at
75 to 80℃, and substantially reduced activities at
lower temperatures.
• At 37℃, Taq polymerase has only about 10% of
its maximal activity.

Taq DNA Polymerase
• Recombinant Taq DNA Polymerase is the enzyme
of choice for most PCR applications.
• The half-life of enzyme is >40 minutes at 95°C.
• The error rate of Taq DNA Polymerase in PCR is
2.2x10-5 errors per nt per cycle;

Other thermostable Polymerases
• In addition to Taq DNA polymerase, several other
thermostable DNA polymerases have been isolated
and expressed from cloned genes. Three of the
most-used polymerases are described in the
following table:
Source and Properties3’-5’
Exonucleases
DNA
Polymerases
From Thermus aquaticus.
Half life at 95℃is 1.6 hours.
NoTaq
From Pyrococcus furiosus.
Appears to have the lowest error rate
of known thermophilic DNA
polymerases.
YesPfu
From Thermococcus litoralis; also
known as Tli polymerase.
Half life at 95 C is approximately 7
hours.
YesVent

The Error Rate
• One of the most discussed characteristics of
thermostable polymerases is their error rate.
• Error rates are measured using several different
assays, and as a result, estimates of error rate
vary, particularly when the assays are performed
by different labs.
The Total Error RateDNA
Polymerases
1 x 10-4 to 2 x 10-5 errors per base pairTaq
appears to have the lowest error rate at
roughly 1.5 x 10-6 error per base pair
Pfu
between Taq and PfuVent

Reliability/Fidelity
Average error rates(mutation frequency/bp/duplication)
increased as follows:
 Pfu (1.3 x 10-6)
 Deep Vent (2.7 x 10-6)
 Vent (2.8 x 10-6)
 Taq (8.0 x 10-6)
 exo- Pfu and UlTma
(approximately 50 x 10-6)

Reference
• Yasumasa Kimura et al. Optimization of turn-back primers in isothermal
amplification (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089485/
• R. Manojkumar and Mrudula Varanat(2006) Polymerase Chain Reaction:
Types and Its Application in theField of Biology. International journal of
tropical medicine 1 (4):156-161
• Voet,D, Voet,J. Biochemistry Vol.1 3rd ed.
• Alberts, Johnson, Lewis. Molecular Biology of The Cell 4th ed.
• Introduction to Plant Biotechnology By- H.S. Chawala
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys
.html
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=146123&rendert
ype=abstract
http://www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm
http://www.fermentas.com/techinfo/pcr/pcrprotocolpfu.htm

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