Sanjana Fatema Chowdhury

2013431012

PCR
 Molecular biology has been revolutionized by pcr
 A method that efficiently amplifies a specific segment of
DNA molecule in logarithmic and controlled fashion.
 Kary mullis conceived pcr in 1983 and received Nobel
Prize for chemistry.
 The chemistry involved in PCR depends on the
complementarity (matching) of the nucleotide bases in
the double-stranded DNA helix. When a molecule of
DNA is sufficiently heated, the hydrogen bonds holding
together the double helix are disrupted and the
molecule separates or denatures into single strands.
 If the DNA solution is allowed to cool, the
complementary base pairs can reform to restore the
original double helix. In order to use PCR, the exact
sequence of nucleotides that flank (lay on either side of)
the area of interest (the target area that needs to be
amplified), must be known.
 This is the absolute minimum data necessary before a
typical PCR reaction can be used. This data is necessary
for the design of PCR primers that are 5'-3'
oligonucleotides of about 20 nucleotides in length.
These are designed to be complementary to the flanking
sequences of the target area

Prerequisite of a PCR
 Primer
 DNTP
1
Sanjana Fatema Chowdhury
2013431012

 Polymerase
 DNA sequence
 Must have ‘known region’ in DNA to be amplified
The Two primers are designed to be complementary.
--Forward primer
--Reverse primer
The two primers (primer pair) can then be synthesized
chemically and will then serve as leaders or initiators of the
replication step.
The key to the replication reaction is that it is driven by a
heat-stable polymerase molecule that reads a template DNA
in the 3’-5’ direction and synthesises a new complementary
template in the 5’-3’ direction, using free dideoxy nucleoside
triphosphates (dNTP’s = nucleotide bases) as building blocks.

PCR Principles
1. Denaturation:
The reaction is started by heating the mixture at 94°C. At this
temperature the hydrogen bonds that put hold together the
two polynucleotides of the double helix are broken, so the
target DNA becomes denatured into single-stranded
molecules. Pcr can be done without extracting DNA from the
bacterial body.
2. Annealing:
The temperature is then reduced to 50-60°C (30-35 sec). This
results in some re-joining of the single strands of the target
DNA, but also allows the primers to attach to the annealing
position.
3. Elongation:

2
Sanjana Fatema Chowdhury
2013431012

The primer binding single stranded DNA solution is heated to

72°C just below the optimum for Taq polymerase. DNA
synthesis can now begin in the presence of heat stable
polymerase, PCR buffers, dNTPs and Mg molecules.
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.
*(Elongation time is specific for specific number base pairs-
30 sec for 500bp)

The cycle of denaturation–annealing–synthesis is now

repeated. In subsequent cycles, the number of short

3
Sanjana Fatema Chowdhury
2013431012

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.
At the end of a PCR a sample of the reaction mixture is
usually analysed by agarose gel electrophoresis, sufficient
DNA having been produced for the amplified fragment to be
visible as a discrete band after staining with ethidium
bromide. This may by itself provide useful information about
the DNA region that has been amplified, or alternatively the
PCR product can be examined by techniques such as DNA
sequencing.

**If elongation time is increased, what will be the loss?

-Energy loss

4
Sanjana Fatema Chowdhury
2013431012

5
Sanjana Fatema Chowdhury
2013431012

The correct
temperature to use:
During each cycle of a PCR, the reaction mixture is
transferred between three temperatures
(Figure 9.7):
l The denaturation temperature, usually 94°C, which breaks
the base pairs and releases single-stranded DNA to act as
templates in the next round of DNA synthesis;
l The hybridization or annealing temperature, at which the
primers attach to the templates;
l The extension temperature, at which DNA synthesis occurs.
This is usually set at 74°C, just below the optimum for Taq
polymerase.
The annealing temperature is the important one because,
again, this can affect the specificity of the reaction. DNA–DNA

6
Sanjana Fatema Chowdhury
2013431012

hybridization is a temperature-dependent phenomenon. If

the temperature is too high no hybridization takes place;
instead the primers and templates remain dissociated (Figure
9.8a). However, if the temperature is too low, mismatched
hybrids—ones in which not all the correct base pairs have
formed—are stable (Figure 9.8b). 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. If
mismatches are tolerated, the number of potential
hybridization sites for each primer is greatly increased, and
amplification is more likely to occur at non-target sites in the
template molecule.

7
Sanjana Fatema Chowdhury
2013431012

Limitations:
One disadvantage of PCR is that errors are introduced into
the amplified DNA copied at low but in ssignificant
frequencies.
Taq polymerase doesn’t contain built in 3’-5’ proof reading
ability and consequently it produces higher than normal
frequency of replication errors.
If an incorrect nucleotide is incorporated during an early pcr
cycle it will be amplified just like any other nucleotide in the
sequence.
Quite high frequency is required
A second disadvantage of taq polymerase is that it amplifies
long pairs of dna rather than a 2000 nucleotide base pair.
If long segmants of DNA need to be amplified the more
processive Tfa poly from thermosfacus is used in case of taq
polymerase.

Primer Design
Primers determine the specificity and efficiency of
amplification in a PCR.
Poor specificity – PCR products with extra unrelated and
undesirable amplicons.
Poor efficiency – less amplification of a product from a
template to the theoretical two fold increase for each PCR
cycle. That’s why Primers are the critical components of PCR.

8
Sanjana Fatema Chowdhury
2013431012

Parameters of primer design

1. The length
2. The terminal nucleotide
3. Reasonable GC content
4. Tm( melting temperature)
Length:
- Longer primer gives more specificity but anneal with
lower efficiency and results a significant decrease in
yield.
- May be require when using eukaryotic genomic DNA.
- Shorter primer anneal very efficiently but may not
give sufficient specificity.
- May be required when amplifying a single template
such as small plasmid.
The terminal nucleotide:
- The 3’ end of the primer should be correctly base
paired to the template otherwise the polymerase will
not be able to state. 3’ end of the primer should be
correctly base paired in order to synthesize more
strand of complementary DNA.
- Having C or G as the 3’ terminal nucleotide makes the
binding of the 3’ end of the primer to the template
more stable.
- The 3’ terminal position in the primer is also essential
for controlling mispairing.
- Three G or C residue in a row near the 3’ end of the
primer should be avoided to minimize nonspecific
primer annealing.

9
Sanjana Fatema Chowdhury
2013431012

- The 3’ end is also important in the prevention of

homologies within a primer pair.
- Primers should not be complementary to each other
particularly at their 3’ end.
- Otherwise will lead to the undesirable primer dimer
- Results in the amplification of the primers themselves
Reasonable GC content:
- PCR primers should maintain a reasonable GC
content.
- GC content should be remain 40-60%
Tm value:
- 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.
- Loss of specificity arises with a lower Tm and primer
with higher Tm has a greater chance of mispriming
under too low temperature.
- If too high temperature is used the primer of the pair
with the lower Tm it will not be associated. ( line sure
na 30.52)
- Tm of the two primer will be within 5°C so that the
primers anneal efficiently at the same temperature.
- Oligonucleotides 20 bases long with a 50% G+C
content generally have Tm values in the range of 56-
62°C.
- The temperature Tm (containing 18 nucleotides or
fewer) is given by the following equation-

10
Sanjana Fatema Chowdhury
2013431012

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.

33 minutes, lecture 9

11
Sanjana Fatema Chowdhury
2013431012

Applications
Because PCR is automated and a typical set of cycles can be
carried out on many separate samples simultaneously in a
few hours, and starting amounts of DNA can be vanishingly
small, PCR has a number of applications where the standard
cloning techniques that we will cover later might be
inappropriate, especially where speed and the number of
samples to be processed are important or where the amount
of DNA available is very limited. Here are some of the
applications.
1. DNA sequencing: PCR in the presence of
dideoxynucleoside triphosphates (ddNTPs), the chain-
terminating inhibitors of DNA synthesis used for DNA
sequencing, allows DNA sequencing reactions to be run
successfully with very small amounts of template. This is
known as cycle sequencing. It requires a specially developed
enzyme that combines the properties needed for sequencing
(especially processivity) with thermostability.

12
Sanjana Fatema Chowdhury
2013431012

2. Diagnostic: PCR is useful as a diagnostic tool, e.g. in the

identification of specific genetic traits or for the detection of
pathogens or food contaminants. One of the first applications
of PCR to genetic diagnosis was for sickle cell anaemia,
allowing analysis to be completed within a day rather than
the weeks taken by the conventional approach of
hybridization analysis of DNA from cells. The sickle cell
mutation in the beta globin gene destroys a restriction site,
and the test involved PCR amplification of this region of the
genome and analysis of the PCR products for the presence or
absence of the restriction site. In general, PCR products can
be analysed by (a) use of restriction enzymes, although it is
rare for a mutation to create or destroy a restriction site; (b)
determining whether an oligonucleotide probe specific for a
particular allele is able to hybridize to PCR products; (c)
electrophoresis, screening for mobility changes caused by
sequence differences between a PCR product carrying a
mutant allele and a wild-type molecule; (d) sequencing the
PCR products directly. Tests for the presence of particular
pathogens can be made using primers specific for the
genomes of those organisms. This permits detection at
extremely low levels. A similar approach can be used to
detect DNA from contaminating sources in food.
3. Forensic: The ability to amplify DNA from regions of the
genome that are highly polymorphic (and, therefore, which
are variable between individuals) starting with samples
containing very small amounts of DNA (e.g. single hairs or
traces of body fluids, such as blood and semen) leads to
applications in forensic work.
A number of polymorphic regions have been used as targets
for amplification, including: the D loop of mitochondrial DNA

13
Sanjana Fatema Chowdhury
2013431012

(mitochondrial DNA also has the advantage of being present

at a high copy number per cell), which is variable between
individuals (much more so than the rest of the mitochondrial
genome); the tandemly repeated minisatellites (also known
as variable numbers of tandem repeats (VNTRs)) and
microsatellites used in conventional genetic fingerprinting;
and human leucocyte antigen
(HLA) sequences. In one of the first widely publicized cases,
the technique was applied in 1991 to the skeletal remains of
a murder victim who had been wrapped in a carpet and
buried from 1981 until the discovery of the bones in 1989.
DNA isolated from these bones was heavily degraded, to
fragments of less than 300 nucleotides, so analysis by
conventional genetic fingerprinting techniques involving
restriction digestion, Southern blotting and hybridization
would not have been possible. The poor condition of the DNA
also made PCR amplification of minisatellites impracticable,
so amplification of shorter microsatellite regions composed
simply of varying numbers of copies of the simple repeated
sequence -CA- was used. PCR products from the victim could
be matched with those from the putative mother and father,
and the provisional identification of the body, which had
been made earlier on the basis of facial reconstruction and
dental records, was confirmed.
4. Present-day population genetics: The ability to amplify
material rapidly from a large number of DNA preparations
leads to applications in population genetics, allowing, for
example, the determination of frequencies of particular
alleles in a large collection of individuals. There are a number
of PCR techniques that provide information from many parts
of the genome simultaneously. One such is random

14
Sanjana Fatema Chowdhury
2013431012

amplification of polymorphic DNA (RAPD) analysis. This uses

relatively short primers that will anneal to many different
sites in the genome under study, producing many different
bands when the PCR products are analysed
electrophoretically. The similarities and differences between
the band patterns generated from genomic DNA of different
organisms provides information on their genetic relatedness.
Another technique involves the amplification of a large set of
restriction fragments of genomic DNA, whose sizes can be
compared electrophoretically. Because of the similarity to
conventional restriction fragment length polymorphism
(RFLP) analysis, this technique is called amplified fragment
length polymorphism (AFLP). PCR is also used in population
genetic studies of microsatellites (or other tandemly
repeated sequences), single nucleotide polymorphisms and
transposons.
A particular advantage of using PCR in population genetic
studies is that, with appropriately designed specific primers,
it may be possible to amplify DNA from one organism that
cannot be separated from others, such as a particular
bacterial strain in a mixed population. (Such primers will
anneal to the target DNA from the organism of interest, but
not to DNA from others.)
This approach, therefore, can be used to study genetics of
bacteria or other organisms that cannot be cultured
axenically.
5. Archaeology and evolution: PCR can be used with old
material as well as more recent samples, and it is often
possible to amplify ancient (less-old material is sometimes
called historic or stale)
DNA from museum specimens and archaeological remains.

15
Sanjana Fatema Chowdhury
2013431012

Multiple copy sequences, such as mitochondrial DNA or

chloroplast DNA, are a particularly useful target, although
nuclear DNA is also retrievable in some cases. Comparison of
polymorphic sequences from the ancient DNA with
sequences observed today allows inferences to be made
about the origins of particular populations or species. For
example, the thylacine or marsupial wolf has been extinct for
some time, although it was once a widespread species in
Australia. Morphological data led to controversy over
whether it was more closely related to a group of South
American marsupial carnivores or to other Australian species
such as the Tasmanian devil. Mitochondrial DNA was
successfully amplified by PCR from skin and museum
specimens collected in the late 19th and early 20th centuries.
The sequence of the amplified DNA was determined, and it
was interpreted as showing that the thylacine was more
closely related to the Australian species than the South
American species, and that the morphological similarities to
the South American species resulted from convergent
evolution.

RT-PCR
(Real Time PCR/Reverse Transcriptase PCR)
In many applications of PCR, the starting template material is
not DNA, but indeed RNA. Examples include PCR assays
designed for the diagnosis off viral infections where the virus
in question has a RNA genome, i.e., HIV, FMDV, rabies and
many others. Since RNA cannot serve as a template for PCR,
reverse transcription is combined with PCR to make

16
Sanjana Fatema Chowdhury
2013431012

RNA into a complementary DNA (cDNA)) suitable for

PCR. The combination of techniques is colloquially referred to
as RT-PCR.
Reverse transcriptase
This enzyme is a RNA dependent DNA polymerase able to
perform the following reactions:
• Synthesizes a DNA strand on an RNA template
• Removes the RNA strand from the DNA: RNA duplex (RNase
H activity)
• Synthesizes a second DNA strand on the DNA template
This enzyme is used in the replication cycle of Retroviruses, a
group of viruses that converts their RNA genomes into DNA
that will then integrate in the genomes of host cells. The
enzyme preparations that are commercially available are
therefore typically of retroviral origin, the most common of
which are avian myeloblastosis virus (AMV) reverse
transcriptase and Moloney murine leukemia virus (M-MuLV)
reverse transcriptase. Tth polymerase, an enzyme isolated
from the bacterium Thermus thermophilus, is a heats table
DNA polymerase with RT activity and can be used in PCR.
Critical factors in RT-PCR
Great care is needed when working with RNA! RNA is much
less stable than DNA and is quickly degraded by RNase
exonucleases. These enzymes are ubiquitous and are also
heat stable and thus very difficult to get rid of. The method
used for the isolation and the protection of the RNA in
question should be designed with this in mind. As a general
rule all water and buffers used in this process are treated
with di-ethyl pyrocarbonate (DEPC) in a process that will
destroy RNase.

17
Sanjana Fatema Chowdhury
2013431012

Gloves are to be worn at all times when working with RNA

and pipettes, consumables and workspace should be
dedicated to RNA work.
Initiation of the RT reaction - Primers
- Oligo (dT) 12-18
This primer will anneal to the endogenous poli (A)+ tail on
the 3' end of eukaryotic mRNA. Such a primer is used in cases
where the PCR is used to randomly clone full-length cDNA of
an organism.
- Hexanucleotide d(N)6
These primers will anneal to mRNA at any complementary
site. Hexanucleotides are commonly used where nothing is
known about the RNA template, or in cases where a
substantial amount of
RNA secondary structure occurs.
- Specific oligonucleotide primer
These primers are designed to target the specific initiation
point in cases where the sequence to be copied is known.
The RT reaction
As a general rule, 1 μg of RNA is enough material for a
successful RT-PCR. The reverse transcription process and the
PCR can be performed in a one-step procedure where all the
components for RT and PCR are present from the start, i.e.,
RNA, primers, dNTP’s, PCR buffer,
RT and Taq enzymes (or an enzyme that can perform both
functions).
Often a two-step procedure is preferred since it offers the
opportunity to better control the assay and to perform a
reaction titration as needed.

18