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1. In Vitro Amplification of DNA
by PCR
Arif Ullah
Ph.D. Zoology
Presented to: Dr. Dil Ara Abbas
Bukhari
Course title: Molecular Biology
Applied

2. Contents
 Introduction
 PCR components
 PCR steps
◦ Denaturation
◦ Annealing
◦ Extension
 PCR types
◦ Nested PCR
◦ RT-PCR

3. 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.

4. 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.

5. 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

6. 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

7. 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

8. 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

9. PCR COMPONENTS
 Water
 10x reaction buffer
 MgCl2
 dNTPs
 Target DNA
 Forward primer
 Reverse primer
 Polymerase enzyme

10. 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

11. 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

12. 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

13. 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

14. 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

15. Steps in PCR reaction

19. Standard thermocycle

20. Visualization of PCR products
 Gel electrophoresis is employed to
check pcr products
 Visualized under x-rays
 Size is determined by comparing with
ladder.

21. 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)

22. 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)

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. 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

31. heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
Thermocyclers

32. Detection of amplification
products
 Gel electrophoresis
 Sequencing of amplified fragment
 Southern blot

33. 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.

34. 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

35. 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.

36. 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.

37. Cont…
B) If extra spurious product bands
present:
 Increase annealing temperature
 Reduce magnesium concentration
 Reduce number of cycles
 Try different enzyme

38. 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

39. 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.).

40. 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.

41. 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.

42. 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/bran
ds/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)