PCR

(Polymerase chain reaction)

BY
POORNA BASURI.P
M.PHARM
IST YEAR
PCR
Polymerase chain reaction (PCR) is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of DNA across
several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
It is an easy, cheap, and reliable way to repeatedly replicate a focused
segment of DNA, a concept which is applicable to numerous fields in modern
biology and related sciences.
Developed in 1983 by Kary Mullis,PCR is now a common and often
indispensable technique used in clinical and research laboratories for a broad
variety of applications.
These include DNA cloning for sequencing, gene cloning and
manipulation, gene mutagenesis; diagnosis and monitoring of hereditary
diseases; amplification of ancient DNA; analysis of genetic fingerprints for
DNA profiling and detection of pathogens in nucleic acid tests for the diagnosis
of infectious diseases.
• The vast majority of PCR methods rely on thermal cycling, which
involves exposing the reactants to cycles of repeated heating and
cooling, permitting different temperature-dependent reactions—
specifically, DNA melting and enzyme-driven DNA replication—to
quickly proceed many times in sequence. Primers (short DNA
fragments) containing sequences complementary to the target region,
along with a DNA polymerase, after which the method is named,
enable selective and repeated amplification.

• As PCR progresses, the DNA generated is itself used as a template for

replication, setting in motion a chain reaction in which the original
DNA template is exponentially amplified. The simplicity of the basic
principle underlying PCR means it can be extensively modified to
perform a wide array of genetic manipulations. PCR is not generally
considered to be a recombinant DNA method, as it does not involve
cutting and pasting DNA, only amplification of existing sequences.
• Almost all PCR applications employ a heat-stable DNA polymerase,
such as Taq polymerase, an enzyme originally isolated from the
thermophilic bacterium Thermus aquaticus. This DNA polymerase
enzymatically assembles a new DNA strand from free nucleotides, the
building blocks of DNA, by using single-stranded DNA as a template
and DNA oligonucleotides (the primers mentioned above) to initiate
DNA synthesis.

• In the first step, the two strands of the DNA double helix are
physically separated at a high temperature in a process called DNA
melting. In the second step, the temperature is lowered and the two
DNA strands become templates for DNA polymerase to selectively
amplify the target DNA. The selectivity of PCR results from the use of
primers that are complementary to the DNA region targeted for
amplification under specific thermal cycling conditions.
Principle of PCR

The PCR involves the primer mediated enzymatic

amplification of DNA. PCR is based on using the ability of
DNA polymerase to synthesize new strand of DNA
complementary to the offered template strand. Primer is
needed because DNA polymerase can add a nucleotide only
onto a preexisting 3′-OH group to add the first nucleotide.
DNA polymerase then elongate its 3 end by adding more
nucleotides to generate an extended region of double stranded
DNA.
Components of PCR
The PCR reaction requires the following components:
• DNA Template : The double stranded DNA (dsDNA) of interest,
separated from the sample.
• DNA Polymerase : Usually a thermostable Taq polymerase that does not
rapidly denature at high temperatures (98°), and can function at a
temperature optimum of about 70°C.
• Oligonucleotide primers : Short pieces of single stranded DNA (often
20-30 base pairs) which are complementary to the 3’ ends of the sense
and anti-sense strands of the target sequence.
• Deoxynucleotide triphosphates : Single units of the bases A, T, G, and
C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and
the building blocks for DNA synthesis.
• Buffer system : Includes magnesium and potassium to provide the
optimal conditions for DNA denaturation and renaturation; also
important for polymerase activity, stability and fidelity.
Procedure of PCR
All the PCR components are mixed together and are taken
through series of 3 major cyclic reactions conducted in an automated,
self-contained thermocycler machine.
• Denaturation :
This step involves heating the reaction mixture to 94°C for 15-30
seconds. During this, the double stranded DNA is denatured to single
strands due to breakage in weak hydrogen bonds.
• Annealing :
The reaction temperature is rapidly lowered to 54-60°C for 20-40
seconds. This allows the primers to bind (anneal) to their
complementary sequence in the template DNA.
• Elongation :
Also known at extension, this step usually occurs at 72-80°C (most
commonly 72°C). In this step, the polymerase enzyme sequentially
adds bases to the 3′ each primer, extending the DNA sequence in the 5′
to 3′ direction. Under optimal conditions, DNA polymerase will add
about 1,000 bp/minute.
With one cycle, a single segment of double-stranded DNA
template is amplified into two separate pieces of double-stranded DNA.
These two pieces are then available for amplification in the next cycle.
As the cycles are repeated, more and more copies are generated and
the number of copies of the template is increased exponentially
Types of PCR
• Real-time PCR
• Quantitative real time PCR (Q-RT PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Multiplex PCR
• Nested PCR
• Long-range PCR
• Single-cell PCR
• Fast-cycling PCR
• Methylation-specific PCR (MSP)
• Hot start PCR
• High-fidelity PCR
• In situ PCR
• Variable Number of Tandem Repeats (VNTR) PCR
• Asymmetric PCR
• Repetitive sequence-based PCR
• Overlap extension PCR
• Assemble PCR
• Intersequence-specific PCR(ISSR)
• Ligation-mediated PCR
• Methylation –specifin PCR
• Miniprimer PCR
• Solid phase PCR
• Touch down PCR, etc
Applications of PCR

Medical Applications:
1. Genetic testing for presence of genetic disease mutations. Eg:
hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism
2. Detection of disease causing genes in suspected parents who act as
carriers.
3. Study of alteration to oncogenes may help in customization of
therapy
4. Can also be used as part of a sensitive test for tissue typing, vital to
organ transplantation genotyping of embryo
5. Helps to monitor the gene in gene therapy
Infectious disease Applications:
1. Analyzing clinical specimens for the presence of infectious agents,
including HIV, hepatitis, malaria, tuberulosis etc.
2. Detection of new virulent subtypes of organism that is responsible for
epidemics.

Forensic Applications:
Can be used as a tool in genetic fingerprinting. This technology
can identify any one person from millions of others in case of : crime
scence, rule out suspects during police investigation, paternity testing
even in case of avaibility of very small amount of specimens ( stains of
blood, semen, hair etc).
Research and Molecular Genetics:
1. In genomic studies: PCR helps to compare the genomes of two
organisms and identify the difference between them.
2. In phylogenetic analysis. Minute quantities of DNA from any source
such a fossilized material, hair, bones, mummified tissues.
3. In study of gene expression analysis, PCR based mutagenesis
4. In Human genome project for aim to complete mapping and
understanding of all genes of human beings.
Selective DNA isolation
PCR allows isolation of DNA fragments from genomic
DNA by selective amplification of a specific region of DNA.
This use of PCR augments many ways, such as generating
hybridization probes for Southern or northern hybridization
and DNA cloning, which require larger amounts of DNA,
representing a specific DNA region. PCR supplies these
techniques with high amounts of pure DNA, enabling analysis
of DNA samples even from very small amounts of starting
material.
 Some PCR 'fingerprints' methods have high discriminative power
and can be used to identify genetic relationships between individuals,
such as parent-child or between siblings, and are used in paternity
testing. This technique may also be used to determine evolutionary
relationships among organisms
Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2)
Child. (3) Mother. The child has inherited some, but not all of the
fingerprint of each of its parents, giving it a new, unique fingerprint.
Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR
can be used to analyze extremely small amounts of sample. This is often
critical for forensic analysis, when only a trace amount of DNA is
available as evidence.
 Quantitative PCR methods allow the estimation of the amount of
a given sequence present in a sample—a technique often applied to
quantitatively determine levels of gene expression. Quantitative PCR is
an established tool for DNA quantification that measures the
accumulation of DNA product after each round of PCR amplification.
Quantitative PCR instrument
A quantitative PCR instrument is a machine that amplifies and
detects DNA. It combines the functions of a thermal cycler and a
fluorimeter, enabling the process of quantitative PCR.
The first quantitative PCR machine was described in 1993, and
two commercial models became available in 1996. By 2009, eighteen
different models were offered by seven different manufacturers. Prices
range from about 20,000 USD to 150,000 USD
Principal performance dimensions of quantitative PCR
instruments are thermal control, fluorimetry and sample throughput.
Thermal control
Efficient performance of quantitative PCR requires rapid, precise,
thermal control.
30 cycles of PCR have been demonstrated in less than 10
minutes. Rapid cycling provides several benefits, including, reduced
time to result, increased system throughput and improved reaction
specificity. In practice however, engineering trade-offs between ease of
use, temperature uniformity, and speed, mean that reaction times are
typically more than 25 minutes.
Thermal non-uniformity during temperature cycling contributes
to variability in PCR and, unfortunately, some thermocyclers do not
meet the specifications claimed by manufacturers.[10] Increasing the
speed of thermal cycling generally reduces thermal uniformity, and can
reduce the precision of quantitative PCR.
 The temperature uniformity also has a direct effect on the ability
to discriminate different PCR products by performing melting point
analysis. In addition to uniformity, the resolution with which
instruments are able to control temperature is a factor which affects their
performance when performing high resolution melting analyses.

Therefore speed, precision and uniformity of thermal control are

important performance characteristics of quantitative PCR instruments.
Fluorimetry
Quantitative PCR instruments monitor the progress of PCR,
and the nature of amplified products, by measuring fluorescence.
The range of different fluorescent labels that can be
monitored, the precision with which they can be measured, and the
ability to discriminate signals from different labels, are relevant
performance characteristics.
By using an instrument with sufficient optical channels and
extensive assay optimisation, up to 7 separate targets can be
simultaneously quantified in a single PCR reaction. However, even
with extensive optimisation, the effective dynamic range of such
multiplex assays is often reduced due to interference between the
constituent reactions.
 The noise in fluorescence measurements affects the precision
of qPCR. It is typically a function of excitation source intensity
variation, detector noise and mechanical noise. Multi factorial
analysis has suggested that the contribution of mechanical noise is
the most important factor, and that systems with no moving parts in
their optical paths are likely to provide improved quantitative
precision.
In addition, when performing high resolution melting
analyses, one factor that affects the sensitivity of heteroduplex
detection is fluorimetric precision.
Therefore the number of optical channels and the level of
noise in fluorescence measurements are also important
performance characteristics of quantitative PCR instruments.
THANK YOU