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.
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 upto 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.
3 Basic Protocol for Polymerase Chain
Reaction
Components
and reagents
A basic PCR set up requires the following essential
components and reagents :
1. Template DNA
containing the DNA region (target) to be amplified.
2. 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).
3. Taq polymerase or other thermostable, high fidelity
DNA polymerase (Pfu polymerase isolated from Pyrococcus furiosus).
4. Deoxyribonucleotide triphosphates (dNTPs), which
are the building-blocks for a newly synthesized DNA strand.
5. Buffer solutions to provide a suitable chemical
condition for optimum activity and stability of the DNA polymerases.
6. Divalent cations (eg. magnesium or manganese ions).
They act as a co-factor for Taq polymerase which increases its polymerase
activity. Generally Mg2+ 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
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
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
thermostable polymerases are used). 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.
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.
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
complimentarity between them. The polymerase enzymes initiate the replication
from 3’ end of the primer towards the 5’end of it.
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.
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.
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 tests
have 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.
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 singlestranded
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.
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.
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