PCR

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PCR Technology
The polymerase chain reaction (PCR): copying (amplifying) specific sequences in DNA
(or in RNA: reverse transcriptase) millions of copies are produced within a few hours.
PCR is a technique analogous to the biosynthesis of DNA (replication) that takes place in
living cells .(DNA amplification)
PCR is used to amplify a sequence of DNA using a pair of oligonucleotide primers each
complementary to one end of the DNA target sequence. These are extended towards
each other by a thermostable DNA polymerase in a reaction cycle of three steps:
denaturation, primer annealing and polymerization.
Characteristics simple, fast, amplification of a target DNA, specificity and sensitivity
General PCR Procedure:
1) Design specific or suitable primers by using manual method or software or from
articles
2) Extraction and purification of DNA from sample
3) PCR mixture (mix template DNA, two appropriate oligonucleotide primers, Taq or
other thermostable DNA polymerases, deoxyribonucleoside triphosphates
(dNTPs), and a buffer in PCR tubes
4) PCR cycle ( the mixture is cycled in thermal cycler many times (usually 30)
through temperatures that permit denaturation, annealing, and synthesis to
exponentially amplify a product of specific size and sequence
5) Analysis of PCR products (amplified DNA): The PCR products are then displayed
on an appropriate gel (agarose , polyacrylamide) , for the PCR product size
expected and examined for yield and specificity. Many important variables can
influence the outcome of PCR.
PCR reaction mixture
(1) DNA templates (2) A pair of primers (3) DNA polymerase (Taq polymerase)
(4) dNTPs (5) Buffer pH, salt, Mg 2+
The PCR cycle:
Initial denaturation
(strand separation)
940
C 3min 1 cycles
Denaturation
(strand separation)
940
C 1min
35 cycles
Primer annealing
(Primer binding)
550
C-630
C 1min
Extension
(DNA synthesis)
720
C 1min
Final extension
(DNA synthesis)
720
C 5min 1 cycles
Storage 40
C Hold

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The basic steps in a PCR experiment are as follows
1. The mixture is heated to 94°C, at which temperature the hydrogen bonds that
hold together the two strands of the double-stranded DNA molecule are broken,
causing the molecule to denature.
2. The mixture is cooled down to 50–60°C. The two strands of each molecule could
join back together at this temperature, but most do not because the mixture
contains a large excess of short DNA molecules, called oligonucleotides or
primers, which anneal to the DNA molecules at specific positions.
Annealing It is critical that the primers anneal stably to the template, because, 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. However, if the
temperature is too low, mismatched hybrids—ones in which not all the correct
base pairs have formed—are stable
3. The temperature is raised to 72°C. This is a good working temperature for the Taq
DNA polymerase that is present in the mixture. The Taq DNA polymerase
attaches to one end of each primer and synthesizes new strands of DNA,
complementary to the template DNA molecules, during this step of the PCR.
4. The temperature is increased back to 94°C. The double-stranded DNA molecules,
each of which consists of one strand of the original molecule and one new strand
of DNA, denature into single strands. This begins a second cycle of denaturation–
annealing–synthesis, at the end of which there are eight DNA strands. By
repeating the cycle 30 times the double-stranded molecule that we began with is
converted into over 130 million new double-stranded molecules, each one a copy
of the region of the starting molecule delineated by the annealing sites of the two
primers.
Figure ( ) the basic steps
in the polymerase chain reaction

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Template: Almost any source that contains one or more intact target DNA molecule can,
in theory, be amplified by PCR, This includes DNA prepared from blood, sperm or any
other tissue, from older forensic specimens, from ancient biological samples or in the
laboratory from bacterial colonies or phage plaques as well as purified DNA. Whatever
the source of template DNA, PCR can only be applied if some sequence information is
known so that primers can be designed.
Enzymes: Thermostable DNA polymerases which have been isolated and cloned from a
number of thermophilic bacteria are used for PCR. ~1kb / min is the rate of DNA
synthesis by this enzyme. The most common is Taq polymerase from Thermus
aquaticus. It survives the denaturation step of 95°C for 1–2 min, having a half-life of more
than 2 h at this temperature. Because it has no associated 3′ to 5′ proofreading
exonuclease activity, Taq polymerase is known to introduce errors when it copies DNA –
roughly one per 250 nt polymerized. For this reason, other thermostable DNA
polymerases with greater accuracy are used for certain applications.
Primers: A pair of oligonucleotides of about 18–30 nt with similar G+C content will serve
as PCR primers as long as they direct DNA synthesis towards one another. The
oligonucleotide primers are the most critical element in terms of successful PCR. 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. One consideration is distance between the primers. Smaller DNA
fragments are amplified more efficiently than longer DNA fragments. The DNA fragment
to be amplified should not be greater than about 3 kb in length and ideally less than 1 kb.
The key to the PCR lies in the design of the primers:
Typical PCR primers are anything between 18-28 nucleotides in length
The G+C composition should ideally be similar to that of the desired amplicon and
should in general be between 50-60%
The calculated Tm for a primer pair should be balanced
A Tm 55°C -72°C is desired (62-65°C is best)
Check for complementarity in 3' ends of primer pairs - this lead to primer - dimer
artifacts
Avoid any significant secondary structure within primers i.e. internal palindromic
sequences
Runs of 3 or more C’s and G’s at 3' ends promote mispriming in G/C rich regions
Palindromic sequences within the primers should be avoided
Avoid an A and especially a T at the 3’ end of a primer (this allow ‘breathing’ in the
hybridisation of the primer to the template)
Avoid any potential mismatches in the 3’end of primers
For short oligonucleotides (<25 nt), the annealing temperature (in °C) can be
calculated using the formula: Tm = 2(A+T) + 4(G+C), where Tm is the melting
temperature and the annealing temperature is approximately 3–5°C lower.
If the DNA sequence being amplified is known, then primer design is relatively easy.
The region to be amplified should be inspected for two suitable sequences of about
20 nt with a similar G+C content, either side of the region to be amplified (e.g. the site
of mutation in certain cancers). If the PCR product is to be cloned, it is sensible to
include the sequence of unique restriction enzyme sites within the 5′-ends of the
primers.

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PCR optimization: PCR reactions are not usually 100% efficient, even when using
cloned DNA and primers of defined sequence. It may be necessary to vary the annealing
temperature and/or the Mg2+
concentration to obtain faithful amplification.
PCR variations:
Nested PCR This can be used when the target sequence is known, but the number of
DNA copies is very small (e.g. a single DNA molecule from a microbial genome), or if the
sample is degraded (e.g. a forensic sample). The process involves two consecutive
‘rounds’ of PCR. The first PCR uses so-called ‘external’ primers, and the second PCR
uses two ‘internal’ (or ‘nested(’ primers that anneal to sequences within the product of the
first PCR. This increases the likelihood of amplification of the target sequences by
selecting for it using different primers during each round. Thus, nested PCR also
increases the specificity of the reaction, since a single set of primers used in isolation
may give a reasonable yield but several bands, while the use of a second set of primers
ensures that a unique sequence is amplified, e.g. in microbial diagnostics.
Inverse PCR
This is a useful technique for amplifying a DNA sequence flanking a region of known
base sequence, e.g. to provide material for characterizing an unknown region of DNA.
The DNA is cut with a restriction enzyme so that both the region of known sequence and
the flanking regions are included. This restriction fragment is then circularized and cut
with a second restriction enzyme with specificity for a region in the known sequence. The
now linear DNA will have part of the known sequence at each terminus, and by using
primers that anneal to these parts of the known sequence, the unknown region can be
amplified by conventional PCR. The product can then be sequenced and characterized.
Reverse transcriptase-PCR (RT-PCR)
This technique is useful for detecting cell-specific gene expression (as evident by the
presence of specific mRNA) when the amount of biological material is limited. Using
either an oligo-dT primer to anneal to the 30 polyadenyl ‘tail’ of the mRNA, or random
hexamer primers, together with reverse transcriptase, cDNA is produced which is then
amplified by PCR. RT-PCR is often a useful method of generating a probe, the identity of
which can be confirmed by sequencing.
Multiplex PCR: multiple pairs of primers are added, PCR can be used to amplify more
than one DNA fragment in the same reaction and these fragments can easily be
distinguished on gels if they are of different lengths. This use of multiple sets of primers is
often used as a quick test to detect the presence of microorganisms that may be
contaminating food or water, or be infecting tissue.
PCR with several targets can be monitored by (target-specific) probes labeled with
different types of fluorescent dye (i.e. dyes with different emission spectra. As multiple
primers are used, extra care is required in order to prevent the formation of primer–
dimers. In some cases, one primer can be shared by two targets. For example, a
sequence in the 16S rRNA gene in Bacteroides forsythus and Prevotella intermedia has
been amplified with one forward primer (a broad-range primer common to both species)
and two species-specific reverse primers. Multiplex PCR has been used e.g. for detecting
mecA and coa genes in Staphylococcus aureus, for diagnostic virology ‫؛‬for detecting
toxin genes in Clostridium difficile and for sub speciation of Campylobacter jejuni isolates.
Multiplex PCR is also used e.g. for amplification of STRs in human DNA profiling (e.g.
CODIS.( An alternative approach is selector-based multiplex PCR.

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PCR Mutagenesis (Site Directed Mutagenesis)
PCR can be used to manipulate DNA. For example, site-directed mutagenesis can be
carried out by designing primers with single nucleotide mismatches. Since the primers
serve as templates in subsequent rounds of DNA replication the PCR products will
contained the introduced nucleotide. Similarly, restriction sites are easily added to the
PCR products for subsequent subcloning.
Touchdown PCR: In a typical reaction, each of the PCR temperature cycles will differ by
1°C in the annealing temperature and each of these cycles is run twice. The range of
temperatures will typically be over 10-20°C (20- 40 cycles) and in the process the
touchdown temperature will have been reached and passed.
This basic approach may of course be changed in terms of temperature range, the
temperature drop and the individual and total numbers of cycles. The rationale of this
method is that preference is given to the reaction with the highest Tm (and therefore the
highest specificity).
Allele-specific PCR: use of an allele-specific primer (and a gene-specific primer) to
amplify a particular allele among a mixture of alleles.
Asymmetric PCR: a procedure in which the concentration of one primer is much lower
than that of the other; it is used for obtaining a particular strand of the template dsDNA.
Real-time PCR
Conventional PCR techniques rely on end-point detection of amplified product, e.g. by
electrophoretic separation and staining. However, such methods are time-consuming and
are only semi-quantitative, since they are based predominantly on the detection of an
amplified fragment (band) in a sample, rather than being designed to give exact
information on its abundance (copy number). Quantitative analysis is only feasible during
the early stages of PCR, where reagents are in excess and where the amount of
amplified product is small, thereby avoiding the problems of product hybridization, which
would compete with primer binding.
Real-time PCR A form of PCR in which it is possible to follow the progress of
amplification – that is, the ongoing increase in numbers of specific amplicons in the
reaction mixture – while it is happening; this approach also permits estimation of the
number of specific target sequences that were present in the reaction mixture before the
beginning of cycling (one form of quantitative PCR), The increase in numbers of
amplicons in a given reaction can be monitored in two main ways: (i) by the use of target
specific probes (which give rise to a fluorescent signal in the presence of specific
amplicons), and (ii) by the use of certain dyes that exhibit dsDNA-dependent
fluorescence, i.e. dyes which fluoresce when they bind to double-stranded DNA (or which
increase fluorescence from a minimal to a significant level when they bind to dsDNA).
During the reaction, fluorescence is recorded cycle by cycle – beginning at cycle 1 and
continuing throughout the reaction to the last cycle (which may be e.g. cycle ~30– 40.( To
estimate the number of target sequences present in the mixture prior to cycling it is
necessary to determine the so called threshold cycle (Ct, or CT), (i.e. the first cycle in
which fluorescence from the reaction exceeds the background fluorescence by a
specified amount) The cycle in which significant fluorescence is first detectable is
inversely proportional to the amount of initial template. A standard curve can be prepared
from this threshold cycle (Ct) and the template concentration of known standards.

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The threshold cycle allows a quantitative estimation of the initial number of target
sequences in the reaction mixture as there is an inverse linear relationship between (i)
threshold cycle and (ii) the logarithm of the number of target sequences prior to cycling.
That is, a linear graph is obtained – over a range of values – if threshold cycle is plotted
against the logarithm of the initial number of target sequences in the reaction mixture.
This relationship can be understood intuitively: the lower the number of target sequences
present in the reaction mixture – prior to cycling – the higher will be the number of cycles
of amplification needed to reach a level of fluorescence corresponding to the threshold
cycle (and vice versa).
Dye fluorescence
This is the simplest and least expensive approach. A fluorescent dye such as SYBR
Green is included in the PCR mixture and the level of fluorescence is monitored as the
reaction proceeds; since SYBR Green binds strongly to dsDNA, showing an
enhancement in fluorescence of over 100-fold, any increase in fluorescence is directly
proportional to the amount of dsDNA produced. Calibration is achieved by running a
series of standards containing known amounts of dsDNA. This approach works well for
optimised single PCR product reactions where non-specific reactions are minimized.
Fluorescent reporter probes
Here a fluorescent reporter dye is covalently bound to the 50 end of an oligonucleotide
probe while a ‘quencher’ group is attached to the 30 end. The probe is designed to
hybridize to an internal region of the target sequence. During PCR, when the DNA
polymerase molecule reaches the hybridized probe, the 50 nuclease activity of the
polymerase will cleave the reporter dye from the rest of the probe, causing an increase in
fluorescence with each cycle that is in direct proportion to the amount of PCR product
being formed, which is itself directly related to the original number of copies of the target
DNA sequence. A commercial example is the TaqMan series of probes – while this
approach is more accurate and reliable than dye fluorescence, it is also far more
expensive, since a specific reporter probe must be synthesised for each target sequence.
Other variants rely on changes in 3D conformation of the probe when it binds to the
target sequence, causing an increase in fluorescence, e.g. Molecular Beacons and
Scorpion probes.

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PCR Applications
o Diagnosis and screening of genetic diseases and cancer
o Rapid detection of slowly growing micro-organisms (e.g. mycobacteria) and
viruses (e.g. HIV)
o Site-Directed Mutagenesis
o Sequencing
o Taxonomy
o Analysis of DNA in archival material
o Gene Expression
o DNA fingerprinting in forensic science
o preparation of nucleic acid probes
o clone screening, mapping and subcloning