polymerase chain reaction PCR technique . Its applications

1. (Polymerase Chain Reaction )
Subject: Advanced Pharmaceutical Analysis (MPA 102T)
Presented By: Shaikh Asif Jamil
Master Of Pharmacy 1st Sem
Department: Pharmaceutical chemistry
Branch: Pharmaceutical analysis

2. INTRODUCTION
Polymerase chain reaction (PCR) is a new, popular molecular biology technique for
enzymatically replicating DNA without using a living organism, such as E. coli or
yeast. The technique allows a small amount of the DNA molecule to be amplified
many times
The word PCR is made up of Polymerase– Taq DNA polymerase + chain– cyclic
reaction + reaction– biological activity. Taq DNA polymerase governed cyclic
reaction is known as PCR.

3. The overall idea of the Polymerase Chain Reaction is to get copies of a DNA or gene we
wish to study. we can’t visualize a few DNAs that is why we need to amplify DNA. After the
isolation of thermostable Taq DNA polymerase, the idea of temperature-dependent
amplification came into the picture.
The PCR- polymerase chain reaction is a temperature-dependent process of DNA
amplification. The machine used in the PCR technique is known as a Thermocycler.
A thermostable Taq DNA polymerase, isolated from the hot water bacteria can synthesize
DNA even at a higher temperature. Using ingredients such as dNTPs and other PCR
enhancers along with Taq, one can synthesize DNA in PCR.

4. HISTORY
In 1993, Mullis was awarded the Nobel prize in Chemistry along with Michael Smith
for his work on PCR. The PCR is commonly carried out in a reaction volume of 10-200
ml in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler.
The thermal cycler heats and cools the reaction tubes to achieve the temperatures
required at each step of the reaction.
Many modern thermal cyclers make use of the Peltier effect, which permits both
heating and cooling of the block holding the PCR tubes simply by reversing the electric
current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for
rapid thermal equilibration.

5. However, the story of PCR was begun when the Taq DNA polymerase was isolated from
the thermostable bacteria. In 1996, Thomas D Brook had discovered the bacteria from
the hot spring of water and named it Thermus aquaticus. Later on, in 1976, Chien et
al., isolated DNA polymerase from Thermus aquaticus named it as Taq DNA polymerase.
Most thermal cyclers have heated lids to prevent condensation at the top of the
reaction tube.
Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction
mixture or a ball of wax inside the tube.
Thermocycler: The machine thermocycler provides various temperatures for each step
to complete. Denaturation, annealing, and extension of DNA occur at different
temperatures thus the machine is known as a thermocycler.

6. The first PCR machine was a series of three different water
baths with three different temperatures. The traditional
machine did not have a digital display or a temperature
controller. In those days, scientists have to transfer PCR tubes
in each water bath manually at least 35 times.
Each water bath had a thermometer for monitoring
temperature. Karry Mullis had achieved PCR amplification
through this process. However, in Year 1985, PerkinElmer
introduced the first automated PCR machine. Because of that
PerkinElmer is one of the pioneers and tech giant companies
in making PCRs.

7. DEFINITION
A common genetic tool- a laboratory technique used to obtain multiple copies of target DNA
fragments using Taq DNA polymerase in a temperature-dependent reaction is called a PCR- a
polymerase chain reaction.”
Theoretically, the definition of the PCR can be as stated,
“PCR is a technique in which using the dNTPs, primers, Taq DNA polymerase, and template DNA,
artificial gene synthesis can be done.”

8. Or we can say, “PCR- a polymerase chain reaction is a cyclic temperature-dependent
reaction used to amplify the gene of interest.”
In short, we can define PCR as,
“An in vitro DNA amplification or synthesis technique is known as PCR.”
Theoretically, the definition of the PCR can be as stated,
“PCR is a technique in which using the dNTPs, primers, Taq DNA polymerase, and template
DNA, artificial gene synthesis can be done.”
Or we can say,
“PCR- a polymerase chain reaction is a cyclic temperature-dependent reaction used to
amplify the gene of interest.”
In short, we can define PCR as,
“An in vitro DNA amplification or synthesis technique is known as PCR

9. PRINCIPLE
The polymerase chain reaction is carried out in a reaction mixture which comprises
the DNA extract (template DNA), Taq polymerase, the primers, and the four
deoxyribonucleoside triphosphates (dNTPs) in excess in a buffer solution.
The tubes containing the mixture reaction are subjected to repetitive temperature
cycles several tens of times in the heating block of a thermal cycler (apparatus which
has an enclosure where the sample tubes are deposited and in which the
temperature can vary, very quickly and precisely, from 0 to 100°C by Peltier effect) .
The apparatus allows the programming of the duration and the succession of the
cycles of temperature steps. Each cycle includes three periods of a few tens of
seconds. The process of the PCR is subdivided into three stages as
1) DENATURATION
2) ANNEALING
3) EXTENSION

11. DENATURATION
The dsDNA becomes single-stranded at a higher temperature
during denaturation. Here hydrogen bonds between two DNA
strands break.
Temperature: 90°C to 95°C
Time 30 sec to 90 sec
In a denaturation two single-stranded DNA form from the
double-stranded one. At 94ºC temperature, the double-
stranded DNA opens up by breaking hydrogen bonds. The
process of denaturation is followed by the initial denaturation
for 5 to 7 minutes at the same temperature.

12. ANNEALING
The primer binds or anneals to its exact complementary
sequence on a DNA during the annealing step. The primer
provides a site for the initiation of synthesis.
The annealing temperature is usually ranging from 55ºC to
65ºC. Annealing temperature lower than that leads to non-
specific bindings while higher temperature leads to
amplification failure. 45 seconds to 1 minute are enough
for the second step, annealing for more than 1 minute
causes non-specific amplification.
Temperature: 55°C to 65°C
Time: 30 to 60 sec
After the denaturation, primer anneals to ssDNA at its exact annealing
temperature. Based on the GC content of primers, every primer has its own
annealing temperature.

13. EXTENSION
Taq DNA polymerase uses the 3’ end of the primer and starts DNA synthesis by
adding nucleotides to the growing DNA strand.
Temperature: 70°C to 72°C
Time: 45 Sec
After the binding of the primer, it’s time to expand the DNA strand. Here in the
extension step the Taq DNA polymerase comes into action and adds dNTPs to the
DNA strand. The temperature for the extension is 72ºC for 45 seconds.

15. REQUIREMENTS
Chemicals:
i) dNTPs(deoxynucleotide triphosphate) :
Deoxynucleotide triphosphates are artificially synthesized nucleotides that bind to the
growing DNA strand. With the help of the Taq DNA polymerase, the dATP, dGTP, dCTP and
dTTP bind at their complementary nucleotides on the growing DNA strand.It is basically
building block of DNA.
ii) Distilled water :
iii) Enzyme Taq DNA polymerase :
The PCR technique is entirely based on the activity of Taq DNA polymerase. If Taq DNA
polymerase was not discovered, the PCR might not be discovered.
Thermostability, the unique property of the Taq makes amplification possible during PCR.
Thermostability means it can work finely at a higher temperature. Notably, no other bodily
enzyme can function at a higher temperature of more than 37ºC.
The Taq DNA polymerase settles at the ssDNA- primer junction and utilizes it as a substrate
for the catalytic reaction. In the final step of extension, using the substrate it starts dNTP
insertion.
1 unit of Taq is sufficient for a 25μL PCR reaction.

16. iv) PCR reaction buffer
PCR enhancers help to boost reaction and amplification efficiency thus PCR buffer is as
important as other ingredients. Additionally, the PCR buffer maintains the constant pH of
the reaction nearly 7.9 to 8.5 by keeping the constant chemical environment for the PCR
reaction.
The pH of the buffer is controlled by the addition of Tris.
Mgcl2, DMSO, KCl, albumin, betaine, BSA, glycerol, (NH4)2SO4, and formamide are some
of the chemicals commonly used in the PCR buffer. The composition of each ingredient
may vary from manufacturer to manufacturer.
However, in each PCR buffer, the MgCl2 must be included because it is worked as a
cofactor for the Taq DNA polymerase.
Three important functions of the MgCl2 in a PCR reaction are,
•To improve or boost the activity of Taq DNA polymerase.
•To facilitate effective and accurately complementary primer binding.
•To increase the melting temperature .
•To increase the amplification efficiency of the reaction.

17. All components are crucial and equally important but the PCR buffer stands out as a
‘kind of reaction booster.’ It has chemicals that help reactions to occur correctly and
that’s why it is required.
Common PCR reaction buffer components are MgCl2, KCl, DMSO, Albumin, (NH4)2 SO4
and various salts. Each ingredient has its own role in the reaction. But it remains
ineffective for some special reactions, for example high GC-rich(guanine-cytosine directly
proportional melting temp) template.
Now talking about some atypical or special reactions like templates with high GC
content, a conventional amplification setup isn’t sufficient enough here. It needs
additives that can effectively amplify such templates.
DMSO is one among many other PCR additives that can help the template with high GC
content .

18. vi) Template DNA:
The template must be DNA only. Plasmid DNA, bacterial DNA, cDNA, or gDNA
can be utilized as a template. The template DNA should be highly purified DNA
that has a purity of around ~1.80 and a quantity of up to 200ng. The DNA
works as a substrate for an enzyme when it is denatured.
The good quality of extracted DNA can boost the resulting efficiency of the
polymerase chain reaction.
Another important PCR ingredient is PCR primers. RNA primer governs the replication
reaction, normally, however, DNA primers are utilized during PCR instead of RNA
primers. The Taq DNA polymerase doesn’t have proof-reading activity thus it can’t
remove RNA primers. Probably, this is the reason, we are using DNA primers in PCR.
v) Primers :
The PCR primers are artificially synthesized oligonucleotide sequences of DNA ranging
from 18 to 22 bases in length, short DNA sequences which anneal at the single-
stranded template DNA at its exact complementary position.

20. Instruments: thermocycler, spinner and agarose gel electrophoresis unit.
vii)Thermocycler/ PCR machine :
The PCR machine is known as a thermocycler. This machine is simply a heating block
(just like our iron) which provides the constant temperature and even rapidly changes
between two temperature states.
The machine has a lower block of metal having deep wells for putting PCR tubes. Also,
the temperature of the inner environment is maintained by the heating block present
on the upper side of the lead.

21. Latest PCR
machine
Further, the machine contains the display, power on and off switch, and cooling
assembly. The machine has the ability to heat and cool the PCR tube in a short period
of time

22. PROCEDURE
Take a sterile PCR tube and start adding reagents as shown
 starts adding reagents in a sequential manner to reduce the chance of
error.
If you have a ready-to-use mastermix, you can add it directly, this will
save time and increases the efficiency of the reaction.
After the completion of reaction preparation, close all the tube caps and
spin it properly, so that all the reagents mix well.
Now put the tubes in the PCR machine one by one in the pre-set PCR
protocol. Remember: don’t waste time setting protocol during the PCR,
set it before the reaction preparation, and immediately run the PCR.
Meanwhile start preparing the gel for agarose gel electrophoresis,
because it will also take time for around 60 to 90 minutes.

23. After completion of the PCR reaction, turn off the machine and collect all the tubes in
an “orderly manner”.
Rest tubes for some time in a freeze before doing agarose gel electrophoresis.
how to interpret the results of PCR in brief,
Run a DNA ladder along with the PCR amplicons so that we can analyze the results.
Based on the migration of DNA fragments in the gel and our in silico PCR or primer 3
results we can assume what size our PCR amplicons are.
See the image below,

24. TYPES
Conventional PCR: The simplest version or the original PCR technique utilizes only a simple Taq DNA
polymerase and no modifications called conventional PCR.
Gradient PCR: Gradient PCR is one of the widely used modifications of native PCR in which for
optimizing the PCR reaction, different temperature gradients are created in a machine. Using these
different temperature gradients, the template DNA amplification efficiency can be checked.
The best annealing temperature can be selected for further consecutive reactions. Besides this, the
efficiency of different PCR enhancers can also be checked at different temperatures using gradient PCR.
Hotstar PCR: The benefit of using the Taq DNA polymerase in the PCR reaction is its stability at a higher
temperature, however, it is also its limitations.
As it is also able to synthesize the DNA at a lower temperature too, using the hot start modifications,
the Taq is inserted only at the time of denaturation. For doing that, different strategies of inactivating
Taq DNA polymerase early in the reaction are available. One of them is the use of enzyme liked
antibody.
At a higher temperature, the antibody released the enzyme in the reaction. However, the main
objective of the hot start is to activate Taq only when the reaction starts.

25. Realtime RT PCR: Yet, another amazing modification of the native PCR is the real-time PCR in which
using the fluorochrome chemistry, the template DNA can be estimated.
A probe attached with the fluorochrome emits fluorescence once it is hydrolyzed from the template
and the template is measured. The amount of fluorescence emitted is directly proportional to the
amount of DNA present in the sample.
This is the principle of real-time PCR which is now widely used in diagnostic and microbial
identification.
Here the catch is the use of the colored molecule, although, different types of probes are used for
different applications.
Reverse transcription PCR: Reverse transcription PCR is actually a variant of real-time PCR in which
instead of DNA the amount of RNA can be measured.
An enzyme called reverse transcriptase converts the total mRNA into the cDNA which is measured
using the same chemistry of the real-time PCR.
Thus the amount of the mRNA present in a sample can be estimated using this type of PCR.
As we know, the total mRNA translates into protein, therefore the gene expression can be measured
using reverse transcription PCR.

26. Immuno PCR:
Immuno PCR is a combination of real-time PCR and ELISA methods. Using the sensitivity
of the ELISA method in the quantification, the specificity of the PCR reaction can be
increased using the Immuno PCR.
In silico PCR: The in silico PCR is a computational tool used to estimate or predict the
results of actual PCR reaction. We have covered an amazing article on a step-wise guide
on how to do in silico PCR
Droplet PCR: The droplet PCR is further, an amazing enhancement of the PCR
quantification in which using the droplet; the amount of the template DNA is estimated
Droplet PCR is an assay used to estimate the amount of the template, especially, for
sensitive assays such as quantification of pathogens.
Asymmetric PCR: The Asymmetric PCR is used to amplify only a single DNA strand for DNA
sequencing and probe hybridization. It needs more PCR cycles, a template-specific
primers and annealing temperature.

27. Nested PCR:
Using one of the nested PCR along with the flanking primers, the efficiency of the PCR
reaction can be increased by employing the nested PCR methods.
Colony PCR: A rapid, high throughput PCR method in which the insert or the plasmid
DNA is amplified directly from the bacterial colony. For that, the bacterial colonies are
taken and PCR is performed directly on it which helps in amplifying insert directly
without extracting plasmid DNA. However, two sets of primers are used for that, one for
plasmid specific and one for amplifying the rest of the DNA.
In situ PCR: In situ-PCR is yet another excellent method for rapid amplification of a
sample DNA. in this method the amplification of target DNA is done directly on the side
or in situ. All the reagents such as dNTPs, primers and PCR buffers are added directly on
the slide to do PCR. This method is widely used for paraffin-embedded tissues or for
formalin-fixed tissues.

28. APPLICATION
Diagnosis of inherited disease: PCR is most routinely used in the diagnosis of some
inherited diseases such as sickle cell anemia, thalassemia, MTHFR gene mutation, etc.
This technique is appropriate for single-gene disorders. The result is 99% accurate as
compared with other methods.
Microbial identification: the microbial culture technique is traditional and time-
consuming and the chance of infection is also high in the case of culturing. In modern
days, PCR is used in the identification of microbes. The bacteria’s unique DNA
sequence is targeted for the identification of particular bacteria. It will give a result
within 3 to 4 hours.
Additionally, PCR is also applicable to the diagnosis of infectious diseases such as HIV
or HPV. Again the method is the same as the identification of microbes. The unique
DNA sequence of a particular virus is targeted for identification.

29. DNA fingerprinting and genetic imprinting: PCR is the first choice for DNA fingerprinting. For
more detail on DNA fingerprinting read the article: DNA fingerprinting
Criminal verification, identification of a person, and material cell contamination can be
detected using DNA fingerprinting.
The PCR is one of the best techniques for marker assistant selection. RFLP, AFP, RAPD, STS,
VNTR, and STR are some of the PCR-based techniques.
PCR is applicable in the prenatal diagnosis of inherited disease as well.
PCR helps in detecting cancer genes and infections.
Further PCR is applicable to sex determination and sex identification.
Apart from mutation detection, PCR is useful in gene expression studies too. The expression of
a particular gene can be measured using RT PCR. It is even applicable in gene cloning.
mRNA studies are also possible due to the reverse transcriptase PCR and we can calculate
gene expression through it.
PCR amplification is one of the important steps in DNA sequencing and microarray.

30. The PCR is also useful in the validation of personalized medicines.
The PCR is used in;
Gene editing Gene manipulation Genetic engineering RNA research DNA and RNA
quantification cDNA and gDNA library preparation Developing new assays
There are many applications of PCR. It is a technique now essential in cellular and
molecular biology.
It permits, especially in a few hours, the “acellular cloning” of a DNA fragment through an
automated system, which usually takes several days with standard techniques of molecular
cloning.
PCR is widely used for diagnostic purposes to detect the presence of a specific DNA
sequence of this or that organism in a biological fluid.
 It is also used to make genetic fingerprints, whether it is the genetic identification of a
person in the context of a judicial inquiry, or the identification of animal varieties, plant, or
microbial for food quality testing, diagnostics, or varietal selection.
 PCR is still essential for performing sequencing or site-directed mutagenesis.
there are variants of PCR such as real-time PCR, competitive PCR, PCR in situ, RT-PCR, etc.

31. LIMITATIONS
Need of target DNA sequencing information:
Primer designing for unexplored ones
Boundary regions of DNA to be amplified must be known
Infidelity of DNA application:
Taq Pol- no proof reading mechanism – error 40% after 20 cycles
Short size and limiting amount of PCR product:
Up to 5kb can be easily amplified.
Up to 40kb can be amplified with some modifications.
Cannot amplify gene >100kb
Cannot be used in genome sequencing project

32. REFERENCE
Polymerase chain reaction – Wikipedia
Polymerase_Chain_Reaction_PCR_A_Short_Review.pdf
Polymerase chain reaction short review by Anwar Khan Modern Medical College
Journal · February 2013
Pelt-Verkuil E, Belkum A, John P. A brief comparison between in vivo DNA
replication and in vitro PCR amplification. Principles and Technical Aspects of
PCR Amplification. Netherlands: Springer; 2008. pp. 9-15
Polymerase Chain Reaction- Definition, Principle, Steps, Procedure, Protocol,
Applications and Types (geneticeducation.co.in)