Polymerase Chain Reaction (PCR)
PCR is a technique used to amply a target DNA. Many copies of DNA segment is prepared in vitro.
This technique was discovered by K.Mullis, he was presented with highest reward the Nobel Prize in 1993.
It is simple and easy technique at practical stages. It is no doubt one of the first approach employed for DNA analysis. In the technique one should be well aware of the base order of target part of DNA, which is going to be amplified. So that this information utilized in making the primers.
Primers are reverse and forward, each is compatible to the 5prime’ end and 3prime’ end respectively to the target segment of DNA sequence.
Furthermore dNTPs, Taq Ploymerase, buffer and some other reagents are required. It is thought that PCR is analogue to the replication process occurs in the cell, as both give the new complementary strands. The making of fresh strand is rely on the already occurring one. It has replaced the traditional DNA cloning techniques since it has the same function, PCR can achieve this in very little interval as compared to the previous one.
Although along with its drawbacks, PCR is marvelous technique which allows the scientists to study the nucleic acids and have a resonance effect on the biological sciences.
In early
days scientists have to mix the polymerase after every cycle because the
polymerase cannot survive the during denaturation step. After that thermostable
polymerase was discovers i.e. Taq polymerase from the bacterium thermos
aquaticus. Thermocycler is used to run the PCR.
Principle
This method relies on cyclic heating of target sequence which allows the repetitive production of the sequence. For the amplification of target sequence, the primers are necessary. As the reaction goes on,
the part of DNA produced is further automatically used for
amplification in the mixture. And the target of DNA is made for many
times.
PCR Machine
How Does it Work?
There are five
major things to be required for this procedure
·
The DNA template which is
to be copied
·
Primers, these are
oligonucleotide sequences, both reverse and forward primers are used.
·
dNTPs for making a fresh
strand of nucleotides
·
Taq polymerase
·
Buffer to control the reaction
conditions
·
A thermocycler machine
·
Cations like potassium or
magnesium
Procedure
All the above thing are mixed in Eppendorf, and then placed in the thermocycler. Then repeated cycles of high temperature and cooler temperature are run after one another. It is done via predefined series of steps.
At starting DNA undergoes denaturation, in which DNA strands are separated at very high temperature. Then the temperature is decreased in the subsequent step, so that the polymerase does it work to anneal the target sequence of DNA. Polymerase does this work very selectively. Such selectivity of polymerase brought about by the complementarity of the primer used for DNA amplification. The condition always remains specific for this thermal cycling.
A PCR
conventionally has around 30-35 cycles and mostly this is in common practice.
The cycle is basically steps of temperature change for a specific period of
time; it has almost 3 steps of temperature change. This procedure is often
start with high temperature around 95oC and then proceeded to
decrease in temperature so that the product gets annealed and extended. At the end storage temperature is applied.
There are different parameter take into consideration, to employ temperature
for a specific time period in each cycle. The concentration of dNTPs and ions
which are used, type of enzymes, and the denaturation temperature
are some of the parameters that are included.
Stages
The stages involves in the PCR
reaction
Ø
Denaturation
Separating
the double strand of DNA, and two single strand are
formed
Ø
Annealing
Temperature
is lowered so that the primers become attach to the separated template region
Ø
Extending
Temperature is raised a bit, thus the taq polymerase joins
the dNTPs to give fresh strand
Details of these Steps
Ø Initiation
The Eppendorf containing the template DNA and other things are placed in thermocycler. This
step includes heating the reaction mixture up to 95oC, as the thermostable
polymerase is present. We does so until around 10 minutes.
Ø Denaturation
They H-bonding
between the two strands diminishes and two strands get separated from each
other. It makes two single stranded DNA. Both the separated strands now work as
template for the primers and both will give fresh strands. Only one thing is
need to ponder about in this step, that temperature should be high enough and
the DNA is placed in this, should remain for such a time that the separation of
strands are ensured. It might take about 30 seconds.
Ø Annealing
Now the reaction mixture should be cooled down up to 50-65oC. This will ensure and allows the
primers to make H-bonding with respective DNA templates so that the process of
fresh strand production may be started. Remembering thing is that, the exact
temperature for annealing relies largely on the denaturation temperature of the used
primers. Usually temp is kept 3-5oC below the denaturation temperature of used primers. The primer length should be around 20-30 base pairs, but best length is 24 base pair primers should be used for this purpose. Primer maybe
a DNA or RNA. Then primers are constructed keeping in mind the complementarity
with the sequence of the DNA which to be copied. No doubt these primers act as
the starting point and helps polymerase i.e. Taq to anneal the rest of the
strand. Once the primers are bind, then polymerase start joining complementary
bases on the strand. The both strands which are separated are complementary and
are contrary in direction, so here is the utilization of reverse and forward
primers come into being. As one is 5-prime’ end to the other is 3-prime’ end.
This stage usually accomplished in about 10-30 seconds. Note that the annealing
temperature is the key in PCR, if this temperature is not adjusted correctly
then the test may go in-vain and gives error.
Ø Extension Stage
The temperature is being
increased to 72oC, which enables the Taq polymerase to add more
dNTPs to the freshly forming strands. And more Nitrogenous bases are added at this step.
As the Taq polymerase is thermophilic enzyme and it works at its maximum on this
temperature. It can also bears temperature of denaturation step. Taq polymerase enzymes add bases by
forming a phosphodiester bond between the 5-prime phosphate of the dNTPs and the
3-prime –OH of the extending strand. Time for extension, is relies on the
fragment length of DNA we want to copy and also on the taq-polymerase. DNA
polymerase attaches bases around 1000/60sec. The results in a fresh DNA strand.
Mentioned 3 steps in the thermocycler are being followed to get copies. Usually cycling around twenty
to forty times gives enough fresh target DNA, to proceed further analysis.
Ø Final Elongation
Temperature here
keeps 70-74oC, for 5 to 15 minutes, after the final cycle, which
give us surety that every dNTP if remains is attached to the extending strand.
Ø Final Hold
This step is
performed for the storage of copied DNA. Temperature here kept around 4-15oC.
Importance of PCR
ü
In
different kind disease diagnosis
ü
Making
copies and to Quantify the DNA
ü
In
forensics
ü
Parental
identification
ü
Mutations analysis
ü
To
know about phylogenetic
ü
Disease
history
ü
Studying
Fossils
ü
Gene
cloning and expression
ü
Vaccines
manufacturing
ü
Creating
mutations
Types of PCR
PCR is highly
versatile technique and can be amended. It customized to get desired
applications in different fields.
Ø Nested PCR
In this
2 pairs of primers are utilized, because there is non-specific amplification of
target DNA, in this case particularity of amplification is increased. 1st
pair primer is utilized to copy DNA; this copied DNA may contains some
nonspecific copying of some DNA parts. Now the 2nd set of primers utilized
in 2nd PCR. This set of primers has different binding sites from the
one which was used in 1st step. This PCR more suitable for the
larger portion of the DNA to copy but obviously the target sequence should be
well known.
Ø Multiplex PCR
This type
of PCR is used to amplify dissimilar DNA sequences of different sizes at
the same time. This will save the time and reaction cost for several times. In
this case multiple primers are used which according to its complementation with
the DNA parts being copied, so multiple genes are copied at once. As multiple
primers are being used so the annealing tem should be optimized so that no
error would occur. The primers we are using should have enough different
lengths so its band width could be easily differentiated from others.
Ø Reverse Transcriptase PCR (RT-PCR)
It is
type of PCR in which mRNA is used. Process of reverse transcription
is done and cDNA is produced. Then this cDNA is used for amplification. Employed
to identify a location of gene. Also gene expressing activity can be determined
by using this. Sequence of a gene also verified by this. Location of introns
and exons are determined if a gene sequence is known. Transcription where it
gets started and terminated these sites could also be located.
Ø Quantitative PCR
To
quantify the target sequence of DNA, it used. It gives the quantities of the DNA, cDNA, or mRNA being used at the starting point. To determine a
sequence in DNA sample its copies, it could be utilized here. Usually probes
which are dyes, fluorescent or luminous are used in quantitative PCR which
tells us how much quantity has been amplified.
Ø Hot Start PCR
During
PCR It is employed to reduce non-specific amplification. In prior to the
addition of taq-polymerase it can be manually performed by heating the mixture
up to 95 degree centigrade. Certain systems for enzymes developed like presence
of antibody or inhibitors bonded by covalent bonds which inhibit the
polymerase’ activity at ambient temperature. Such hybrid polymerases are
developed which are nonfunctional at ambient temp and working at prolonged
temperatures also known as Hot start and Cold finish PCR.
Ø Asymmetric PCR
When the
amplification of only one of the two strands is necessitated then this method is
employed. It amplifies one DNA strands rather than both. When sequence probing
is required this technique is used. Involves routine PCR but the strand whose
amplification is required its primers used in large number. Reason is that
extra PCR cycles are required due to the slow amplification process after the
usage of limiting primer. A latest modification known LATE-PCR, here the
limiting primer is used with high tm than the excessive one, so concentration
of limiting primer will be reduced in mid of reaction.
Ø Nanoparticle-Assisted PCR (nanoPCR)
Efficiency
of the PCR could be enhanced by using some NPs, may be performing better than
real PCR. QDs can also improve specificity of PCR. Single Welled Carbon
Nanotubes and MWCN are used to efficient amplification of larger segments of
the DNA. To improve the efficiency of longer PCR CNPs are also used. ZnO, TiO2, and Ag NPs are widely
used for this purpose. For nanoPCR very high possibility that this technology is
going to improve and development of products.
Ø Allele Specific PCR
This
is based on SNVs cloning technique. The information about the sequence is required
before proceeding, also difference between alleles, and the primers which are
being used and their 3prime ends incorporated with SNVs. When there is mismatch
present between Primer and template PCR amplification is not mush efficient,
here employees the particular SNVs sequence for successful amplification with
an SNV.
Ø Inverse PCR
When
the identification of the flanked sequence near to any genomic inserts is
required this technique is employed. It involves self-ligation, and digestion
of DNA series. Thus on the either side of not known sequences, known sequences
are resulted.
Ø Colony PCR
When a
bacterial colony having a plasmid of our desires gene is required to screened
this method is used. Culturing and purification of the plasmid is not necessary.
Ø In situ PCR
This
process is in fact employed when a PCR is runs in the cell placed in the slide
in vitro. And could seen similar as the hybridization.
Ø AFLP-PCR
Genomic DNA is digested with restriction enzymes, and then sticky ends are produced to these sticky ends adapters are attached, and then from these restricted fragments are chosen for undertaking of amplification, this is AFLP-PCR works. Primers, complementary to the sequence, and a few Nitrogenous bases are used for the selection of the fragment. Then these amplified fragments can be seen by Polyacrylamide Gel Electrophoresis (PAGE) using different probes.
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