PCR is a polymerase chain reaction in which target DNA gets amplified. There are various modifications to PCR reaction to increase sensitivity and specificity such as touchdown PCR, Real time PCR, Hot start PCR, RT-PCR, Colony PCR and asymmetric PCR.

1. Different types of PCR
techniques
Module III
MSc Biotech Part I

2. PCR and its Principle
● PCR also known as “Polymerase Chain Reaction” is a amplification of specific
region of DNA.
● Any region of any DNA molecule can be chosen, so long as the sequences at the
borders of the region are known.
● In order to carry out a PCR, two short oligonucleotides must hybridize to the DNA
molecule, one to each strand of the double helix. These oligonucleotides, which act
as primers for the DNA synthesis reactions, delimit the region that will be amplified.

3. Fig: Hybridization of the
oligonucleotide primers to the
template DNA at the beginning of
a PCR.
1. Denaturation and hybridization of primers

4. 2. Requirements for Amplification:
Amplification is usually carried out by the DNA polymerase I enzyme from Thermus
aquaticus., this organism lives in hot springs, and many of its enzymes, including Taq
polymerase, are thermostable, meaning that they are resistant to denaturation by heat
treatment.
The thermostability of Taq polymerase is an essential requirement in PCR
methodology.
To carry out a PCR experiment, the target DNA is mixed with Taq polymerase, the two
oligonucleotide primers, and a supply of nucleotides. The amount of target DNA can be
very small because PCR is extremely sensitive and will work with just a single starting
molecule.

5. The PCR Reaction
Step I: Denaturation (94°C)
Step II: Annealing (50–60°C)
Step III: Synthesis (74°C)

6. ● The reaction is started by heating the mixture to 94°C. At this temperature the
hydrogen bonds that hold together the two polynucleotides of the double helix are
broken, so the target DNA becomes denatured into single-stranded molecules.
● The temperature is then reduced to 50–60°C, which results in some rejoining of the
single strands of the target DNA, but also allows the primers to attach to their
annealing positions.
● DNA synthesis can now begin, so the temperature is raised to 74°C, just below
the optimum for Taq polymerase. In this first stage of the PCR, a set of “long
products” is synthesized from each strand of the target DNA. These polynucleotides
have identical 5′ ends but random 3′ ends, the latter representing positions where
DNA synthesis terminates by chance.

7. DNA
Synthesis
Synthesis of Short Products

8. ● The cycle of denaturation–annealing–synthesis is now repeated.
● The long products denature and the four resulting strands are copied during the DNA
synthesis stage. This gives four double-stranded molecules, two of which are identical
to the long products from the first cycle and two of which are made entirely of new
DNA.
● During the third cycle, the latter give rise to “short products”, the 5′ and 3′ ends of
which are both set by the primer annealing positions.
● In subsequent cycles, the number of short products accumulates in an exponential
fashion (doubling during each cycle) until one of the components of the reaction
becomes depleted. This means that after 30 cycles, there will be over 130 million short
products derived from each starting molecule.
● In real terms, this equates to several micrograms of PCR product from a few
nanograms or less of target DNA

9. Fig: Theoretical PCR
amplifications of target fragment
with increasing number of cycles

10. Fig: A typical temperature
profile for a PCR

11. Primer Designing
● Primers are the short oligonucleotide sequence which are complementary to
specific region of target DNA.
● The primers are the key to the success or failure of a PCR experiment.
● If the primers are designed correctly the experiment results in amplification of a
single DNA fragment, corresponding to the target region of the template molecule.
● If the primers are incorrectly designed the experiment will fail, possibly because no
amplification occurs, or possibly because the wrong fragment, or more than one
fragment, is amplified.

12. Fig: The results of PCRs with well
designed and poorly designed primers.
Lane 1 shows a single amplified fragment
of the expected size, the result of a well
designed experiment.
In lane 2 there is no amplification
product, suggesting that one or both of the
primers were unable to hybridize to the
template DNA.
Lanes 3 and 4 show, respectively, an
amplification product of the wrong size,
and a mixture of products (the correct
product plus two wrong ones); both results
are due to hybridization of one or both of
the primers to non-target sites on the
template DNA molecule.

13. Direction of Primers:
Primers must correspond with the sequences
flanking the target region on the template
molecule. Each primer must, of course, be
complementary (not identical) to its template
strand in order for hybridization to occur, and
the 3′ ends of the hybridized primers should
point toward one another
Fig: A pair of primers designed to
amplify the human α1-globin gene. The
exons of the gene are shown as closed
boxes, the introns as open boxes.

14. Calculating Number of target sites for particular length of primer
Formula:3,200,000 kb (Size of human genome)/4n
4 Number of dinucleotides
n= Length of primer
For example: If length of the primer is 10 nucleotides then primer will bind once
every 410 base pairs. i.e. once every 1,048,576 base pairs. So, total number of
target sites for a 10 mer primer will be 3,200,000 kb/1048.576 kb= 3051 sites.
Examples: Calculate the target sites for a 12 mer and 15 mer primers.

15. Length of Primers
If the primers are too short they might hybridize to non-target sites and give undesired
amplification products.
To illustrate this point, imagine that total human DNA is used in a PCR experiment with a
pair of primers eight nucleotides in length (in PCR jargon, these are called “8-mers”).
The likely result is that a number of different fragments will be amplified. This is because
attachment sites for these primers are expected to occur, on average, once every 48=
65,536 bp, giving approximately 49,000 possible sites in the 3,200,000 kb of nucleotide
sequence that makes up the human genome.
This means that it would be very unlikely that a pair of 8-mer primers would give a single,
specific amplification product with human DNA

16. ● If a 17 mer primer is used The expected frequency of a 17-mer sequence is once
every 417= 17,179,869,184 bp. This figure is over five times greater than the length of
the human genome, so a 17-mer primer would be expected to have just one
hybridization site in total human DNA. A pair of 17-mer primers should therefore
give a single, specific amplification product.
● The length of the primer influences the rate at which it hybridizes to the template
DNA, long primers hybridizing at a slower rate. The efficiency of the PCR,
measured by the number of amplified molecules produced during the experiment, is
therefore reduced if the primers are too long, as complete hybridization to the
template molecules cannot occur in the time allowed during the reaction cycle. In
practice, primers longer than 30-mer are rarely used.

17. The lengths of the primers
are critical for the
specificity of the PCR.

18. Effect of temperature on Primer Annealing
The annealing temperature is the important one
because, again, this can affect the specificity of the
reaction.
DNA–DNA hybridization is a temperature-
dependent phenomenon.
If the temperature is too high no hybridization
takes place; instead the primers and templates
remain dissociated (Figure a). However, if the
temperature is too low, mismatched hybrids—
ones in which not all the correct base pairs have
formed—are stable (Figure b). If this occurs the
earlier calculations regarding the
appropriate lengths for the primers become
irrelevant, as these calculations assumed that only
perfect primer–template hybrids are able to form.

19. Ideal annealing temperature
● The ideal annealing temperature must be low enough to enable hybridization between
primer and template, but high enough to prevent mismatched hybrids from forming.
● This temperature can be estimated by determining the melting temperature or Tm of
the primer–template hybrid.
● The Tm is the temperature at which the correctly base-paired hybrid dissociates
(“melts”). A temperature 1–2°C below this should be low enough to allow the
correct primer–template hybrid to form, but too high for a hybrid with a single
mismatch to be stable.

20. Calculating Tm
Calculate Tm of following primer
sequences:
1. 5’GAATTAACACCGTT3’
2. 5’TATACCGGTCGAATT3
’
The Tm can be determined experimentally but is more usually calculated from the
simple formula:
Tm = (4 × [G + C]) + (2 × [A + T])°C
in which [G + C] is the number of G and C nucleotides in the primer sequence, and
[A + T] is the number of A and T nucleotides.

21. Fidelity of taq polymerase
● All DNA polymerases make mistakes during DNA synthesis, occasionally inserting
an incorrect nucleotide into the growing DNA strand.
● Most polymerases, however, are able to rectify these errors by reversing over the
mistake and resynthesizing the correct sequence. This property is referred to as the
“proofreading” function and depends on the polymerase possessing a 3′to
5′exonuclease activity.
● Taq polymerase lacks a proofreading activity and as a result is unable to correct
its errors. This means that the DNA synthesized by Taq polymerase is not always
an accurate copy of the template molecule.
● The error rate has been estimated at one mistake for every 9000 nucleotides of DNA
that is synthesized, which might appear to be almost insignificant but which translates
to one error in every 300bp for the PCR products obtained after 30 cycles.

22. ● This is because PCR involves copies being made of copies of copies, so the
polymerase-induced errors gradually accumulate, the fragments produced at the end
of a PCR containing copies of earlier errors together with any new errors introduced
during the final round of synthesis.
● For many applications this high error rate does not present a problem. In particular,
sequencing of a PCR product provides the correct sequence of the template,even
though the PCR products contain the errors introduced by Taq polymerase. This is
because the errors are distributed randomly, so for every molecule that has an error at
a particular nucleotide position, there will be many molecules with the correct
sequence. In this context the error rate is indeed insignificant.

23. When error prone PCR products get
cloned: This is not the case if the PCR
products are cloned. Each resulting clone
contains multiple copies of a single
amplified fragment, so the cloned DNA
does not necessarily have the same
sequence as the original template molecule
used in the PCR. This possibility lends an
uncertainty to all experiments carried out
with cloned PCR products and dictates that,
whenever possible, the amplified DNA
should be studied directly rather than being
cloned.

24. Thermostable DNA polymerases with proofreading activity

25. Types of PCR
● Real time PCR
● Reverse-transcription PCR/RT-PCR
● Multiplex PCR
● Nested PCR
● Touchdown PCR
● Hot start PCR
● Colony PCR

26. Real time PCR
● There is a quantitative relationship between the amount of starting material (target
sequence) and the amount of PCR product at any given cycle.
● It is a modification of the standard PCR technique in which synthesis of the product
is measured over time, as the PCR proceeds through its series of cycles.
● Thus, Quantitative PCR (Q-PCR) is used to measure the amount of PCR product
in real time.

27. There are two ways of following product synthesis in real time:
1. A dye that gives a fluorescent signal when it binds to double-stranded DNA can be
included in the PCR mixture. This method measures the total amount of double-stranded
DNA in the PCR at any particular time, which may overestimate the actual amount of the
product because sometimes the primers anneal to one another in various non-specific
ways, increasing the amount of double-stranded DNA that is present.
2. A short oligonucleotide called a reporter probe, which gives a fluorescent signal
when it hybridizes to the PCR product, can be used. Because the probe only
hybridizes to the PCR product, this method is less prone to inaccuracies caused by
primer-primer annealing. Each probe molecule has pair of labels.

28. Hybridization of a reporter probe to its
target DNA
A fluorescent dye is attached to one end of the
oligonucleotide, and a quenching compound,
which inhibits the fluorescent signal, is
attached to the other end.
Normally there is no fluorescence because the
oligonucleotide is designed in such a way that
its two ends base pair to one another, placing
the quencher next to the dye.
Hybridization between the oligonucleotide
and the PCR product disrupts this base
pairing, moving the quencher away from the
dye and enabling the fluorescent signal to be
generated.

29. Taq Man Probes

30. Taq Man Probes Mechanism:
● This probes used are oligonucleotides with a reporter fluorescent dye attached at
the 5′ end and a quencher dye at the 3′ end.
● While the probe is intact,the proximity of the quencher reduces the fluorescence
emitted by the reporter dye.
● If the target sequence is present, the probe anneals downstream from one of the
primer sites.
● As the primer is extended, the probe is cleaved by the 5′nuclease activity of the
Taq polymerase. This cleavage of the probe separates the reporter dye from the
quencher dye, thereby increasing the reporter-dye signal.
● Cleavage removes the probe from the target strand, allowing primer extension to
continue to the end of the template strand.
● Additional reporter-dye molecules are cleaved from their respective probes with
each cycle, effecting an increase in fluorescence intensity proportional to the
amount of amplicon produced.

31. Schematic representation of a typical
DNA amplification plot obtained using
real-time PCR
● First there is an early background
phase when the fluorescence does
not rise above the baseline.
● In the second stage sufficient
product has accumulated to be
detected above the background and
the fluorescence increases
exponentially.
● In practice, a fixed fluorescence threshold is set above the baseline and the parameter
CT (threshold cycle) is defined as the fractional cycle number at which the
fluorescence passes the fixed threshold.

32. Once CT is reached, the amplification reaction is described by the equation:
Tn = T0(E)n
where Tn is the amount of target sequence at cycle n, T0 is the initial amount of target, and
E is the efficiency of amplification.
In the final stage the reaction efficiency declines until no more product accumulates.
The value of CT will depend on the starting concentration of target DNA: the lower
the initial concentration the more cycles will be required to reach the threshold the higher
the initial concentration less number of cycles will be required to reach threshold.

33. Quantification by real-time PCR. The graph
shows product synthesis during three PCRs, each
with a different amount of starting DNA. During
a PCR, product accumulates exponentially, the
amount present at any particular cycle
proportional to the amount of starting DNA.
The blue curve is therefore the PCR with the
greatest amount of starting DNA, and the green
curve is the one with the least starting DNA. If
the amounts of starting DNA in these three PCRs
are known, then the amount in a test PCR can be
quantified by comparison with these controls.
In practice, the comparison is made by identifying
the cycle at which product synthesis moves above
a threshold amount, indicated by the horizontal
line on the graph

34. RT-PCR (Reverse transcription PCR)
● Reverse transcription (RT)-PCR is
used to amplify RNA targets.
● The RNA template is converted into
complementary (c)DNA by the
enzyme reverse transcriptase.
● The cDNA serves later as a template
for exponential amplification using
PCR.

35. Types of RT-PCR
One step RT-PCR Two step RT-
PCR
● One-step RT-PCR combines the
RT reaction and PCR reaction
in the same tube.
● Only sequence-specific primers
may be used.
● They are easier to set up and
ideal for high throughput
screening.
● The synthesized cDNA is
transferred into a second tube
for PCR.
● Oligo (dT), random hexamer or
gene-specific primers can be
used.
● They are ideal for detection of
several messages from a single
RNA sample.

36. Multiplex PCR
● In Multiplex PCR, two or more primer sets designed for amplification of
different targets are included in the same PCR reaction. Using this technique,
more than one target sequence in a clinical specimen can be amplified in a single
tube.
● The primers used in multiplex reactions must be selected carefully to have similar
annealing temperatures and must be not complementary to each other.
● The amplicon sizes should be different enough to form distinct bands when
visualized by gel electrophoresis.
● Multiplex PCR can be designed in either single-template PCR reaction that uses
several sets of primers to amplify specific regions within a template, or multiple-
template PCR reaction, which uses multiple templates and several primer sets in the
same reaction tube.

37. Overview of Multiplex PCR

38. ● Although the use of multiplex PCR can reduce costs and time to
simultaneously detect two, three, or more pathogens in a specimen, multiplex
PCR is more complicated to develop and often is less sensitive than single-
primer-set PCR.
● The advantage of multiplex PCR is that a set of primers can be used as internal
control, so that we can eliminate the possibility of false positives or negatives.
Furthermore, multiplex PCR can save costly polymerase and template in
short supply.

39. Touchdown PCR
● “Touchdown polymerase chain reaction (PCR)” is a method to decrease off-target
priming and hence to increase the specificity of PCRs.
● The key principle in TD-PCR is to, begin first with an annealing temperature
above the projected Tm, then transitioning to a lower, more permissive
temperature over the course of 10–15 cycles.
● In touchdown PCR the temperature selected for the annealing step is initially set 5˚C–
10˚C higher than the calculated Tm of the primers.
● Annealing under conditions of high stringency favors the formation of perfect
primer–template hybrids. In subsequent cycles, the annealing temperature is
gradually decreased by a small amount so that by the end of the PCR, the annealing
temperature is 2˚C–5˚C below the calculated Tm of the primers.
● By then, the target sequence will have undergone several cycles of geometric
amplification and therefore becomes the dominant product of the PCR.

40. Touchdown PCR

41. ● The use of touchdown PCR is essential when the sequence of the primer might not
match that of the target—for example, if the sequence of the primer has been deduced
from amino acid sequences, when the template DNA may contain several closely
related targets, or when the target DNA is of a different species from that used to
design the primers.
● Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs,
increasing specificity, sensitivity and yield, without the need for lengthy
optimizations and/or the redesigning of primers.
● TD-PCR is particularly useful for templates that are difficult to amplify but can
also be standardly used to enhance specificity and product formation. The procedure
takes between 90 and 120 min, depending on the template length.

42. Hot Start PCR
● This technique is based on the principle of initiating the polymerase reaction at or
above the primer annealing temperature, thereby preempting possible extensions
of the primers nonspecifically annealed at ambient temperature (20°-55°C) at which
Taq Pol retains partial activity.
● For some applications, hot-start PCR may be the only way to amplify successfully
the desired product(s) with high yield and specificity.
● Method I: a hot-start PCR condition can be attained by adding the polymerase (or
any other essential reagent like dNTPs or Mg2+) only when the mixture has
reached a temperature higher than Tm and then starting cycles of primer
annealing and extension.

43. ● Method II: The use of a wax pellet (e.g., AmpliWax of Perkin-Elmer), which can
provide at room temperature a solid-phase barrier between the polymerase and the rest
of the reaction components, has made it substantially easier and safer to perform the
technique in an uninterrupted PCR operation.
● Method III: Introduction of thermolabile antibodies which reversibly block the
polymerase provides an alternative efficient technique for hot-start PCR: as the
thermal cycling starts and temperature of the complete PCR mixture is raised, the
inhibitory antibody is selectively denatured (within 30 sec at >85°C), thereby releasing
fully active Taq Pol that initiates the polymerization reaction under high stringency
priming conditions.(Note: One such antibody, with a trade name of TaqStart, is
commercially available from Clontech.)

44. Hot Start PCR

45. Nested PCR
● Nested PCR is a modification of PCR that was designed to improve sensitivity and
specificity.
● Nested PCR involves the use of two primer sets and two successive PCR
reactions. The first set of primers is designed to anneal to sequences upstream from
the second set of primers and are used in an initial PCR reaction.
● Amplicon resulting from the first PCR reaction is used as a template for a
second set of primers and a second amplification step.
● Sensitivity and specificity of DNA amplification is significantly enhanced with this
technique.
● However, the potential for carryover contamination of the reaction is typically
increased due to additional manipulation of amplicon products.
● To minimize carryover, different parts of the process are physically separated from
one another.

46. Nested PCR

47. Asymmetric PCR
● In an asymmetric PCR, the reaction
preferentially amplifies one DNA strand in
a double-stranded DNA template.
● Thus it is useful when amplification of only
one of the two complementary strands is
needed such as in sequencing and
hybridization probing.
● The whole PCR process is similar to regular
PCR, except that the amount of primer for
the targeted strand is much more than that
of the non-targeted strand.

48. ● As the asymmetric PCR progresses, the lower concentration limiting primer is
quantitatively incorporated into newly synthesized double stranded DNA and
used up. Consequently, linear synthesis of the targeted single DNA strand from the
excess primer are formed after depletion of the limiting primer.
● Asymmetric PCR is not widely used because it has low reaction efficiency and it is
hard to optimize the proper primer ratios, the amounts of starting material, and the
number of amplification cycles.
● Limiting the concentration of one primer lowers its melting temperature below
the reaction annealing temperature. Recently, this process has been changed to be
known as Linear-After-The –Exponential-PCR (LATE-PCR) where the limiting
lower concentration primer has a higher melting temperature than the higher
concentration primer to maintain reaction efficiency.

49. Colony PCR
● Colony or whole cell PCR is the direct PCR amplification of target sequences
from cells without prior isolation or purification of DNA.
● Colony PCR is possible if enough cells lyse as a consequence of the high temperature
in the initial template denaturation step alone or in combination with extra procedures
to make DNA more accessible.
● The advantage of colony PCR over using purified DNA is mainly speed and cost,
as the time consuming DNA extraction step is omitted; but omitting DNA
purification and thereby minimizing sample handling can also increase sensitivity if
the starting material is limiting, as it might be in, for example, forensic applications.

50. ● Very low amounts of starting material may prohibit DNA purification as all
purification procedures are associated with a loss. Less sample handling also lowers
the risk of cross contamination of samples, an important point since PCR is a
sensitive technique, which may be prone to false positive detection.
● The most common application of colony PCR in genetic engineering is probably the
amplification of ligation product sequences within Escherichia coli transformants
after cut and paste cloning.
● The key steps to colony PCR are:
1) Design primers to detect the presence of your insert
2) Set up a standard PCR reaction (primers, dNTPs, polymerase) using the supernatant of
lysed bacteria as template
3) Run your PCR product on a gel to analyze product size.

51. Designing colony PCR primers
The first and perhaps most important
step to colony PCR is designing
primers. There are 3 strategies for
primer design:
1) Insert-specific primers
2) backbone-specific primers
3) orientation-specific primers.

52. 1. Insert-specific primers: Insert-specific primers are designed to anneal to an insert-
specific sequence. This is a “yes or no” kind of test, with a positive clone amplifying
a product and a negative clone resulting in no product. Additionally, this type of
primer only tells you if your specific insert is present but not if it’s in the correct
orientation or even if it’s in your plasmid backbone.
2. Backbone-specific primers: A second option is to design backbone-specific primers.
These primers are designed to anneal to sites that flank the insert site. A positive
clone will produce a larger size product then a negative clone without the insert. This
type of primer pair can tell you if the insert is the correct size and whether it’s within
your backbone.

53. This type of primer pair is also great for screening clones created with the same backbone
but that contain different inserts. When you design primers to anneal outside the cloning
site, it doesn’t matter what the sequence of the insert is, allowing you to use the same
primer pair to screen for the presence of many different inserts. The downside: this type of
primer does not provide information about the orientation of the insert.
3. Orientation-specific primers: If you need information about insert orientation, then
you might consider designing orientation-specific primers. Blunt end cloning is an
example of when you might want to know the orientation of the insert. One member of
this type of primer pair anneals to a sequence flanking the insert and one primer anneals to
the insert. A simple way to create this type of primer pair is to mix-and-match insert-
specific and back-bone specific primers.

54. Analyzing PCR Products of colony PCR