PCR or Polymerase Chain Reaction
• Technique used in molecular biology to create several copies of a certain DNA
segment.
• This technique was developed in 1983 by Kary Mullis, an American biochemist.
• PCR has made it possible to generate millions of copies of a small segment of
DNA.
• This tool is commonly used in the molecular biology and biotechnology labs.
Principle of PCR
• Based on the enzymatic replication of DNA.
• In PCR, a short segment of DNA is amplified using primer mediated enzymes.
• DNA Polymerase synthesises new strands of DNA complementary to the template
DNA.
• The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only.
Therefore, a primer is required.
• Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase.
Components Of PCR
1.DNA Template– The DNA of interest from the sample.
2.DNA Polymerase– Taq Polymerase is used. It is thermostable and does not denature at
very high temperatures.
3.Oligonucleotide Primers- These are the short stretches of single-stranded DNA
complementary to the 3’ ends of sense and anti-sense strands.
4.Deoxyribonucleotide triphosphate– These provide energy for polymerization and are
the building blocks for the synthesis of DNA. These are single units of bases.
5.Buffer System– Magnesium and Potassium provide optimum conditions for DNA
denaturation and renaturation. It is also important for fidelity, polymerase activity, and
stability.
Types of PCR
Real-time In this type, the DNA amplification is detected in real-time with the help
PCR of a fluorescent reporter.
Nested PCR This was designed to improve sensitivity and specificity.
Multiplex This is used for the amplification of multiple targets in a single PCR
PCR experiment.
It amplifies many different DNA sequences simultaneously.
Quantitative It uses the DNA amplification linearity to detect, characterize and quantify
PCR a known sequence in a sample.
Arbitrary It is a DNA fingerprinting technique based on PCR.
Primed PCR It uses primers the DNA sequence of which is chosen arbitrarily
PCR Steps
The PCR involves 3 major cyclic reactions:
A. DENATURATION
• Denaturation occurs when the reaction mixture is heated to 94℃ for about 0.5 to 2
minutes.
• This breaks the hydrogen bonds between the two strands of DNA and converts it into a
single-stranded DNA.
• The single strands now act as a template for the production of new strands of DNA.
• The temperature should be provided for a longer time to ensure the separation of the
two strands.
B. ANNEALING
• The reaction temperature is lowered to 54-60℃ for around 20-40 seconds.
• Here, the primers bind to their complementary sequences on the template DNA.
• Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length.
• They serve as the starting point for the synthesis of DNA.
• The two separated strands run in the opposite direction and consequently there are
two primers- a forward primer and a reverse primer
C. ELONGATION
• At this step, the temperature is raised to 72-80℃.
• The bases are added to the 3’ end of the primer by the Taq polymerase enzyme.
• This elongates the DNA in the 5’ to 3’ direction.
• The DNA polymerase adds about 1000bp/minute under optimum conditions.
• Taq Polymerase can tolerate very high temperatures.
• It attaches to the primer and adds DNA bases to the single strand.
As a result, a double-stranded DNA molecule is obtained.
These 3 steps are repeated 20-40 times in order to obtain a number of sequences of DNA
of interest in a very short time period.
LIMITATIONS
1. Prior information about the target sequence is necessary in order to generate the primers that
will allow its selective amplification
2. Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in
the PCR fragments that are generated
3. Another limitation of PCR is that even the smallest amount of contaminating DNA can be
amplified, resulting in misleading or ambiguous results.
To minimize the chance of contamination, investigators should reserve separate rooms for reagent
preparation, the PCR, and analysis of product.
Reagents should be dispensed into single-use aliquots. Pipettors with disposable plungers and extra-
long pipette tips should be routinely used
Benefits of PCR
• The benefit is that PCR mainly deals with different sectors such as the reputation cycle of
“Denaturation”, “Amplification”, and “Replication” in the segmentation of DNA.
• Specific DNA helps to indicate the presence of microorganisms that “Inhibit bacterial
translation”.
• PCR throughout the field of molecular biology helps researchers to clone and sequence genes
for mutation.
• In recent mobility, a small sample is required for the analysis, PCR is highly sensitive compared
to culture and staining.
• The ability to test antimicrobial resistance, it quickly performs the activity in 4-8 hrs, more cost-
effective for select use than cultural and staining.
• Increase ability to detect fewer organisms such as viruses.
Electrophoresis of PCR-amplified DNA
fragments:
1.Father
2.Child
3.Mother
The child has inherited some, but not all, of the
fingerprints of each of its parents, giving it a
new, unique fingerprint.
Applications of PCR
1. It helps to analyse clinical specimens for infectious agents that include HIV, malaria,
anthrax
2. It provides information to patients regarding resistance to therapy. It is observed that
many cancers are characterised by creating genes that are identified by PCR.
3. It used to identify mutations that occur in many genetic diseases.
4. In a forensic laboratory, PCR is used to identify tiny amounts of DNA obtained from
droplets of blood.
5. A technique allows the generation of large amounts of DNA to purify tiny amounts
of the template and study a particular gene.
6. PCR helps to identify relationships among fields of evolutionary biology. In
“Anthropology”, it is used to understand human migration patterns. In
“Archaeology” used to spot the human race.
Applications of PCR
Medicine Forensic Science Research and Genetics
Testing of genetic Used as a tool in genetic Compare the genome of two
disease mutations. fingerprinting organisms in genomic studies.
Monitoring the gene in Identifying the criminal In the phylogenetic analysis of
gene therapy. from millions of people. DNA from any source such as
fossils.
Detecting disease- Paternity tests Analysis of gene expression.
causing genes in the
parents.
Gene Mapping
