Polymerase chain reaction (PCR) and its applications

Introduction:
Polymerase chain reaction (PCR) is a widely employed technique in molecular biology to
amplify single or a few copies of DNA, generating millions of copies of a particular DNA
sequence. The polymerase chain reaction results in the selective amplification of a target
region of a DNA or RNA molecule. PCR has been extensively exploited in cloning, target
detection, sequencing etc. The method consists of thermal cycles of repeated heating
followed by cooling of the reaction mixture to achieve melting and primer hybridization to
enable enzymatic replication of the DNA.

History:
By 1971, a “repair synthesis" process was reported which was an artificial system
containing primers and templates that can allow DNA polymerase to copy target gene. The
DNA polymerases initially employed for in vitro experiments were unable to withstand
these high temperatures. In 1976, Chien et al discovered a novel DNA polymerase from
the extreme thermophile Thermus aquaticus which naturally dwell in hot water spring (122
to 176 °F). The enzyme was named as Taq DNA polymerase which is stable up to 95°C.
In 1985, Kary Mullis invented a process Polymerase Chain Reaction (PCR) using the
thermo-stable Taq polymerase for which he was awarded Nobel Prize in 1993.
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Basic Protocol for Polymerase Chain Reaction:
1- Components and reagents:
A basic PCR set up requires the following essential components and reagents :

a. Template DNA containing the DNA region (target) to be amplified.

b. Primers that are complementary to the 5' ends of each of the sense (Forward
primer) and anti-sense strand of the DNA target (Reverse primer).
c. Taq polymerase or other thermo stable, high fidelity DNA polymerase (Pfu
polymerase isolated from Pyrococcus furiosus).
d. Deoxyribonucleotide triphosphates (dNTPs), which are the building-blocks for a
newly synthesized DNA strand.
e. Buffer solutions to provide a suitable chemical condition for optimum activity and
stability of the DNA polymerases.
f. Divalent cations (eg. magnesium or manganese ions). They act as a co-factor for
Taq polymerase which increases its polymerase activity. Generally Mg 2+ is used,
but Mn2+ can be applied to achieve PCR-mediated DNA mutagenesis. This is
because higher Mn2+ concentration leads to higher error rate during DNA
synthesis.

Buffer solutions
Divalent cations

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2- Procedure:
Typically, PCR is designed of 20-40 repeated thermal cycles, with each cycle consisting
of 3 discrete temperature steps: denaturation, annealing and extension. The thermal
cycles are often proceeded by a temperature at a high range (>90°C), and followed by
final product extension or brief storage at 4 degree celsius. In PCR cycles, the
temperatures and the duration of each cycle is determined based on various parameters
like the type of DNA polymerase used, the melting temperature (Tm) of the primers,
concentration of divalent ions and dNTPs in the reaction etc. The various steps involved
are:-

a) Initial Denaturation
b) Denaturation
c) Annealing
d) Extension
e) Final extension

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a) Initial denaturation:
Initial denaturation involves heating of the reaction to a temperature of 94–96 °C for
7-10 minutes (or 98 °C if extremely thermo stable polymerases are used). For specifically
engineered DNA polymerases (Hot start Taq polymerases) activity requires higher range
of temperature. The initial heating for such a long duration also helps in gradual and
proper unfolding of the genomic DNA and subsequent denaturation, and thus exposing
target DNA sequence to the corresponding primers.

b) Denaturation:
Denaturation requires heating the reaction mixture to 94–98 °C for 20–30 seconds. It
results in melting of the DNA template by disrupting the hydrogen bonds between
complementary bases, yielding single-stranded DNA molecules.

c) Annealing:
Following the separation of the two strands of DNA during denaturation, the temperature
of the reaction mix is lowered to 50–65 °C for 20–50 seconds to allow annealing of the
primers to the single-stranded DNA templates. Typically the annealing temperature
should be about 3-5 °C below the Tm of the primers. Stable complimentary binding are
only formed between the primer sequence and the template when there is a high sequence
complementarity between them. The polymerase enzymes initiate the replication from 3’
end of the primer towards the 5’end of it.

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d) Extension/Elongation:
Extension/elongation step includes addition of dNTPs to the 3’ end of primer with the
help of DNA polymerase enzyme. The type of DNA polymerase applied in the reaction
determines the optimum extension temperature at this step. DNA polymerase synthesizes
a new DNA strand complementary to its template strand by addition of dNTPs,
condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of
the nascent (extending) DNA strand. Conventionally, at its optimum temperature, DNA
polymerase can add up to a thousand bases per minute. The amount of DNA target is
exponentially amplified under the optimum condition of elongation step. The drawback
of Taq polymerase is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease
proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides.

e) Final elongation & Hold:

Final elongation step is occasionally performed for 5–15 minutes at a temperature of
70–74 °C after the last PCR cycle to ensure amplification of any remaining single-
stranded DNA. Final hold step at 4 °C may be done for short-term storage of the reaction
mixture.
After around 30 cycles of denaturation, annealing and extension, there will be over a
billion fragments that contain only your target sequence. This will yield a solution of
nearly pure target sequence.To check the desired PCR amplification of the target DNA
fragment (also sometimes referred to as the amplicon or amplimer), agarose gel
electrophoresis is employed for separation of the PCR products based on size.
determination of size(s) of PCR products is performed by comparing with a DNA ladder,
which contains DNA fragments of known size, run on the gel along side the PCR
products.

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Applications

1- Infectious disease diagnosis, progression and response to therapy

PCR technology facilitates the detection of DNA or RNA of pathogenic organisms and,
as such, helps in clinical diagnostic tests for a range of infectious agents like viruses,
bacteria,protozoa etc. These PCR-based tests have numerous advantages over
conventional antibody-based diagnostic methods that determine the body's immune
response to a pathogen. In particular, PCR-based tests are competent to detect the
presence of pathogenic agents in-advance than serologically-based methods, as patients
can take weeks to develop antibodies against an contagious agent. PCR-based testshave
been developed to enumerate the amount of virus in a person's blood (‘viral load') thereby
allowing physicians to check their patients' disease progression and response to therapy.
This has incredible potential for improving the clinical management of diseases caused
by viral infection, including AIDS and hepatitis, assessment of viral load throughout and
after therapy.

PCR-based diagnostics tests are available for detecting and/or quantifying a

number of pathogens, including:

1. HIV-1, which causes AIDS

2. Hepatitis B and Cviruses, might lead to liver cancer

3. Human Papillomavirus, might cause cervical cancer

4. Chlamydia trachomatis, might lead to infertility in women

5. Neisseria gonorrhoeae, might lead to pelvic inflammatory disease in women

6. Cytomegalovirus, might cause life threatening disease in transplant patients and other
immunocompromised people, including HIV-1/AIDS patients

7. Mycobacterium tuberculosis, which in its active state causes tuberculosis and can lead
to tissue damage of infected organs.

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2- Diagnosis of genetic diseases
The use of PCR in diagnosing genetic diseases, whether due to innate genetic changes or
as a result of a natural genetic mutations, is becoming more common. Abnormality can
be diagnosed even prior to birth. Single-strand conformation polymorphism (SSCP), or
single-strand chain polymorphism, is defined as conformational difference of single-
stranded nucleotide sequences of identical length as induced by differences in the
sequences under certain experimental conditions. These days, SSCP is most applicable
as a diagnostic tool in molecular biology. It can be used in genotyping to detect
homozygous individuals of different allelic states, as well as heterozygous individuals
who inherit genetic aberrations.

3- Genetic counselling
Genetic counselling is done for the parents to check the account of genetic disease
beforehand to make a decision on having children. This is of course governed by national
laws and guidelines. Detection of genetic disease before implantation of an embryo in
IVF (In vitro fertilisation) also known as pre-implantation diagnosis can also be done
exploiting PCR based method. Further to diagnose inherited or a spontaneous disease,
either symptomatic or asymptomatic (because of family history like Duchene muscular
dystrophy) PCR based method is very useful.

4- Forensic sciences
Genetic fingerprint is one of the most exploited application of PCR (also known as DNA
profiling).Profiles of specific stretches of DNA are used in genetic fingerprinting
(generally 13 loci are compared) which is differ from person to person. PCR also plays a
role in analysis of genomic or mitochondrial DNA, in which investigators used samples
from hair shafts and bones when other samples are not accessible.

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5- Research in Molecular Biology
PCR is an essential technique in cloning procedure which allows generation of large
amounts of pure DNA from tiny amount of template strand and further study of a
particular gene. Some alterations to the PCR protocol can generate mutations (general or
site-directed) in a sequence either by an inserted fragment or base alteration. PCR is used
for sequence-tagged sites (STSs) as an indicator that a particular segment of a genome is
present in a particular clone. A common application of Real-time PCR is the study of
expression patterns of genes during different developmental stages. PCR can also
investigate ‘ON or OFF” of particular genes at different stages in tissues (or even in
individual cells).

6- Others
PCR has numerous applications in various fields. The Human Genome Project (HGP) for
determining the sequence of the 3 billion base pairs in the human genome, relied heavily
on PCR.The genes associated with a variety of diseases have been identified using PCR.
For example, Duchenne muscular dystrophy,which is caused by the mutation of a gene,
identified by a PCR technique called Multiplex PCR. PCR can help to study for DNA
from various organisms such as viruses or bacteria. PCR has been used to identify and to
explore relationships among species in the field of evolutionary biology. In anthropology,
it is also used to understand the ancient human migration patterns. In archaeology, it has
been used to spot the ancient human race. PCR commonly used by Paleontologists to
amplify DNA from extinct species or cryopreserved fossils of millions years and thus can
be further studied to elucidate on.

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