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PCR Lecture

The document provides an overview of the polymerase chain reaction (PCR) technique. It describes the basic components and steps of PCR, including DNA denaturation, primer annealing, and fragment elongation. It also discusses various PCR applications in medical diagnosis, infectious disease detection, forensics, and research. PCR is a powerful technique for amplifying specific DNA regions, enabling various downstream applications.

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Noor Khan
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© © All Rights Reserved
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Download as PPT, PDF, TXT or read online on Scribd
221 views

PCR Lecture

The document provides an overview of the polymerase chain reaction (PCR) technique. It describes the basic components and steps of PCR, including DNA denaturation, primer annealing, and fragment elongation. It also discusses various PCR applications in medical diagnosis, infectious disease detection, forensics, and research. PCR is a powerful technique for amplifying specific DNA regions, enabling various downstream applications.

Uploaded by

Noor Khan
Copyright
© © All Rights Reserved
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
You are on page 1/ 42

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)

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