Polymerase Chain Reaction

Introduction
Advanced molecular technology has become a crucial tool for identifying new genes with
importance in medicine, agriculture, animal production, health, environment, industry other
related areas. Among the applications of molecular techniques is important to highlight the use
of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral,
bacterial, parasitic and fungal agents.

PCR is a process used in molecular biology to amplify a single copy or a few copies of a piece
of DNA across several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence. Mechanisms involved in this methodology are similar to those
occurring in vivo during DNA replication .

Definition of PCR
It is a genetic technique that occurs in vitro which allows the enzymatic synthesis of large
quantities (amplification) of a targeted region of DNA in exponential manner. DNA is synthesized
in the same manner as that seen in vivo (in the cells) using a DNA polymerase (enzymes that cells
use to replicate their DNA) .

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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

Types of PCR
• Conventional (Qualitative)PCR.

• Multiplex PCR.

• Nested PCR.

• RT-PCR and qRT-PCR.

• Quantitative PCR.

• Hot-start PCR.

• Touchdown PCR.

• Assembly PCR.

• Colony PCR.

• Methylation-specific PCR.

• LAMP assay.

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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

Uses of PCR
→ Medicine: detecting infectious organisms, discovering variations and mutations in
genes.
→ Genome Projects: DNA sequencing
→ The law: Genetic fingerprinting
→ Evolutionary biology: taxonomic classification
→ Zoology: research on animal behaviour
→ Ecology: studies on seed dispersal, reducing illegal trade in endangered species,
monitoring release of GMOs
→ Archaeology and palaeontology: ancient DNA, analyzing genetic variations in
animals and plants .

Advantages of PCR

1. Simple technique .
2. Sensitivity .
3. Specificity
4. Fast technique .
5. Versatility .

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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

Applications of PCR

1. Detecting pathogens using genome-specific primer pairs in clinical samples

2.Detection of viral pathogens and other microorganisms which persist in
low levels in infected cells and are difficult to be identified by routine methods
such as HIV or HPV.

3.Diagnosis of genetic disorders such as hemophilia, sickle cell anemia

and thalassemia.
4. Identification of genetic mutations like deletions, insertions and point mutations.
5. Screening specific genes for unknown mutations.
6.Identification and analysis of mutations
in eukaryotic DNA.
7. Gene polymorphisms.
8. Gene expression
9. Forensic Odontology

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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

Basic Protocol for Polymerase

Chain Reaction

A/ 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.Deoxy ribonucleotide 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.
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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

B/ 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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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

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). 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.

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 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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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

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.

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.

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 Medical Laboratory Techniques Department
Title :- PCR
Assist lect. Ola Abbas Khdhair
[email protected]

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), agarose gel
electrophoresis is employed for separation of the PCR products based on size. The
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 alongside the
PCR products.

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