In Vitro Amplification of DNA
by PCR
Arif Ullah
Ph.D. Zoology
Presented to: Dr. Dil Ara Abbas
Bukhari
Course title: Molecular Biology
Applied
Contents
Introduction
PCR components
PCR steps
◦ Denaturation
◦ Annealing
◦ Extension
PCR types
◦ Nested PCR
◦ RT-PCR
Polymerase Chain Reaction
(PCR)
Technique widely used in molecular biology to make multiple
copies of a specific DNA segment.
In vitro technique for amplification of a region of DNA
whose sequence is known or which lies between two regions
of known sequence.
Short history of PCR
1983: Dr. Kary Mullis developed PCR
1985: First publication of PCR by Cetus Corporation
appears in Science.
1986: Purified Taq polymerase first used in PCR
1988: PerkinElmer introduces the automated thermal cycler.
1993: Dr. Kary Mullis shares Nobel Prize in Chemistry for
conceiving PCR technology.
Application
PCR is molecular technique to amplify segment of DNA
Used in clinical and research laboratories for a broad
range of applications
◦ Diagnosis of genetic diseases
◦ Genetic fingerprints
◦ Detection and diagnosis of infectious diseases
◦ Detection of infection in the environment
◦ PCR in research
Principles of PCR
Based on DNA replication in vivo
DNA is unwound to single strand, duplicated, rewound
Amplify DNA in short period of time
Amplify DNA fragment between 0.1 to 10kbp
Some allow to amplify up to 40 kbp
Basic requirements for PCR reaction
A DNA template: DNA target region to amplify.
Size of template can be <0.1 to few kilobase
Total amount of DNA for PCR is 0.05-0.1ug
A DNA polymerase: An enzyme that polymerizes new
DNA strands; heat-resistant Taq polymerase is especially
common, as it is more likely to remain intact during the
high-temperature DNA denaturation process.
Primers:
16-30 nucleotides long primers are used
Complementary to the 3' ends of each of the sense and anti-
sense strands of the DNA target;
without primers there is no double-stranded initiation site at
which the polymerase can bind
Cont…
Deoxynucleotide triphosphates (dNTPs): Building blocks
to synthesizes a new DNA strand.
Reaction Buffer: Providing a suitable chemical environment
for optimum activity and stability of the DNA polymerase.
Bivalent cations: Mg++
Mg ion stimulate polymerase activity
Increase melting temperature of primer
Monovalent cations: Potassium (K) ions
PCR COMPONENTS
Water
10x reaction buffer
MgCl2
dNTPs
Target DNA
Forward primer
Reverse primer
Polymerase enzyme
Dream Taq Green PCR Master Mix
Optimum PCR components are directly
used in form of mastermix
Contain
1. Dream Taq DNA polymerase
2. Optimized Dream Taq Green buffer
3. MgCl2
4. dNTPs
Higher yields compare to conventional Taq
DNA polymerase
Significance of master mix
Direct loading of PCR prodcuts on gel
High yield and high sensitivity of PCR
Amplification of long targets upto 6kb
from genomic DNA and up to 20 kb from
viral DNA
Incorporate modified nucleotides, but
doesnot incorporate dUTP
Procedure of PCR
Carried in reaction volume of 10-200ul in
small tubes called PCR tubes
PCR tubes 0.2-0.5mL
Placed in thermocycler
Main steps
Consists of series of 20-40 repeated temperature
changes called cycles
If 100% efficiency is assumed in each cycle there will
be 220 fold amplification after 20 cycle of pcr
Steps in PCR
Steps Temp. Duration
Denaturation 92°C to 95°C 1min
Annealing 50°C to 55°C 45sec
Elongation 70°C to 75°C 1-2min
Steps in PCR reaction
Standard thermocycle
Visualization of PCR products
Gel electrophoresis is employed to check
pcr products
Visualized under x-rays
Size is determined by comparing with
ladder.
Types of PCR
Allele-specific PCR
Assembly PCR
Asymmetric PCR
Convective PCR
Dial-out PCR
Digital PCR (d PCR)
Hot start PCR
In silico PCR (digital PCR, virtual PCR, electronic
PCR, e-PCR)
Cont…
Intersequence-specific PCR (ISSR)
Methylation-specific PCR (MSP)
Multiplex-PCR
Nanoparticle-Assisted PCR (nanoPCR)
Nested PCR
quantitative PCR (qPCR)
Reverse Transcription PCR (RT-PCR)
Touchdown PCR (Step-down PCR)
Allele-specific PCR
A PCR application in which alleles that differ by one
or more nucleotides can be distinguished on the basis
of PCR amplification.
It permits the detection of any mutation in human DNA
by analysing the PCR products directly in an ethidium
bromide-stained agarose or polyacrylamide gel.
Used in DNA-based diagnostic techniques involving
the diagnosis of genetic and infectious diseases.
Hot start PCR
Modified form of PCR that reduces non-
specific amplification during the initial set up
stages of the PCR.
It may be performed manually by heating the
reaction components to the denaturation
temperature (e.g., 95 °C) before adding the
polymerase.
Multiplex-PCR
Consists of multiple primer sets within a single
PCR mixture to produce amplicons of varying
sizes that are specific to different DNA
sequences.
Annealing temperatures for each of the primer
sets must be optimized to work correctly within
a single reaction.
Nested PCR
Increases the specificity of DNA amplification, by reducing
background due to non-specific amplification of DNA.
Two sets of primers are used in two successive PCRs:
In the first reaction, one pair of primers is used to generate
DNA products, which besides the intended target, may
still consist of non-specifically amplified DNA fragments.
Cont…
The product(s) are then used in a second PCR with a set
of primers whose binding sites are completely or partially
different from and located 3' of each of the primers used
in the first reaction.
Nested PCR is often more successful in specifically
amplifying long DNA fragments than conventional PCR,
but it requires more detailed knowledge of the target
sequences.
Quantitative PCR (qPCR)/Real
Time PCR
Used to measure the quantity of a target sequence
(commonly in real-time).
It quantitatively measures starting amounts of DNA,
cDNA, or RNA. quantitative PCR is commonly used to
determine whether a DNA sequence is present in a
sample and the number of its copies in the sample.
Quantitative PCR has a very high degree of precision.
Reverse transcription PCR (RT-
PCR)
It is used for amplifying DNA from RNA.
Reverse transcriptase reverse transcribes RNA into cDNA,
which is then amplified by PCR.
RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify the
sequence of RNA transcript.
Touchdown PCR (step-down PCR)
A variant of PCR that aims to reduce nonspecific
background by gradually lowering the annealing
temperature as PCR cycling progresses.
The annealing temperature at the initial cycles is
usually a few degrees (3–5 °C) above the Tm of the
primers used, while at the later cycles, it is a few
degrees (3–5 °C) below the primer Tm.
The higher temperatures give greater specificity for
primer binding, and the lower temperatures permit
more efficient amplification from the specific
products formed during the initial cycles.
Thermocyclers
heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
Detection of amplification
products
Gel electrophoresis
Sequencing of amplified fragment
Southern blot
Advantages of PCR
Fairly simple to understand and to use.
Produce Automated, fast, reliable results.
Highly sensitive.
Potential to produce millions to billions of copies of a
specific product for sequencing, cloning, and analysis.
Broad uses.
Defined, easy to follow protocol.
Limitations of PCR
Smallest amount of contaminated DNA can be amplified,
resulting in misleading or ambiguous results.
Need for target DNA sequence information
Boundary regions of DNA to be amplified must be
known.
Infidelity of DNA replication: Taq Pol – no Proof
reading– Error 40% after 20 cycles
Cont…
Short size and limiting amounts of PCR
product
Up to 5kb can be easily amplified .
Up to 40kb can be amplified with some modifications.
Cannot amplify gene >100kb
Cannot be used in genome sequencing projects.
Things to try if PCR does not
work
A) If no product ( of correct size )
produced:
Check DNA quality.
Reduce annealing temperature.
Increase magnesium concentration.
Add dimethyl sulphoxide ( DMSO ) to assay ( at
around 10% ).
Use different thermostable enzyme.
Throw out primers - make new stocks.
Cont…
B) If extra spurious product bands
present:
Increase annealing temperature
Reduce magnesium concentration
Reduce number of cycles
Try different enzyme
Applications of PCR
Common applications of PCR in various fields can be
explained in following categories:
Medical Applications
Infectious disease Applications
Forensic Applications
Research and Molecular Genetics
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 scene.
•rule out suspects during police investigation.
•paternity testing even in case of availability of
very small amount of specimens (stains of blood,
semen, hair etc.).
Research and molecular genetics
In genomic studies: to compare the genomes of two
organisms and identify the difference between them.
In phylogenetic analysis: minute quantities of DNA
from any source like a fossilized material, hair, bones,
mummified tissues.
In study of gene expression analysis, PCR based
mutagenesis.
In Human genome project for aim to complete
mapping and understanding of all genes of human beings.
Conclusion
PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA for STD detection,
but it is also important for genetic predisposing for
defects.
The PCR technology can also be employed in law
enforcement, genetic testing of animal stocks and
vegetable hybrids, and drug screening along with
many more areas.
References
Molecular Cell Biology ( Lodish, Darnell..)
https://sciencebasedmedicine.org/wp-content/
uploads/2011/09/nested-pcr.gif
http://11e.devbio.com/ch/03/wt/031005/figure1.jpg
https://www.thermofisher.com/pk/en/home/brands/
thermo-scientific/molecular-biology/molecular-
biology-learning-center/molecular-biology-
resource-library/basic-principles-rt-qpcr.html
Paul, N. (2010). Hot start PCR. Methods in
Molecular Biology. 630. pp. 301–318
Fundamentals of Biochem ( Voet, Voet, Pratt)
