Introduction to PCR.pptx

An introduction to Central Dogma of Molecular
Biology and Polymerase Chain Reaction
XX

Structure of DNA
Source - https://www.genome.gov/genetics-glossary/Deoxyribonucleic-Acid

https://twitter.com/BMCBiology/status/920603314978476033
Rosalind Franklin -
Best known for her contributions to the discovery of the
molecular structure of deoxyribonucleic acid (DNA)
(https://www.britannica.com/biography/Rosalind-Franklin)
James Watson and Francis Crick with a model of DNA Helix
(https://www.sciencephoto.com/media/914842/view/watson-and-crick-with-
their-dna-model)

David Baltimore – Discovered reverse Transcriptase
(https://nationalmedals.org/laureate/david-baltimore/) Stanley Prusiner – Discovered Prions
(https://www.ucsf.edu/news/2014/07/116151/prusiner
-appointed-board-foundation-food-and-agricultural-
research)

Central Dogma of Molecular Biology
• It was proposed by Francis Crick in 1957 – first published in 1958
• It states that genetic information flows in one direction - Deoxy-Ribonucleic Acid (DNA) is converted to
Ribonucleic Acid (RNA) by Transcription, which is converted to Protein by Translation.
• Most pathogens (including DNA Viruses) follow the Central Dogma, while RNA Viruses and Retroviruses follow
the Modified Central Dogma
Fig. Modified Central Dogma
(https://web.northeastern.edu/bbarbiellini/CBIO3580/DOGMA/DNA_
CenDog.html)

Diagrammatic Representation of Central Dogma (https://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology)

Fig. Transcription and Translation (https://www.cancer.gov/publications/dictionaries/cancer-terms/def/transcription)

Equipment used in PCR
Freezer (-20 °C or -80 °C)
• Used for long term storage of samples
• Equipped with adjustable shelves to accommodate various types of sample containers
• Also has programmable thermostat and temperature monitoring systems
Refrigerated Centrifuge
• High speed centrifuge
• Used for nucleic acid extraction
Class II A2 Biological safety cabinet
• Used in sample processing
• Provides personnel, product and environmental protection through filtered, laminar and unidirectional airflow
• Air is filtered through High Efficiency Particulate Air (HEPA) Filter, and cabinet is maintained at negative
pressure
PCR Workstation
• Workspace enclosed on three sides, provides the space for preparation of master mix

Thermal Cycler
• Used to amplify segments of DNA via the Polymerase Chain Reaction
• Thermal block - tubes holding the reaction mixtures can be inserted
• Cycler - raises and lowers the temperature of the block in pre-programmed steps
Spectrophotometer
• Analytical instrument used for the objective calculation of visible light, UV light, or infrared light emission or
reflection
• Used to verify the purity of extracted DNA sample
• Absorbance Ratio between 260nm/280nm is used for this purpose
Real time PCR Machine
• Thermal cycler equipped with an optical detection module to measure the fluorescence signal

PCR Workstation
Thermal Cycler

Figure showing principle of Spectrophotometer
(https://microbenotes.com/spectrophotometer-principle-instrumentation-applications/)
Spectrophotometer
(https://www.unicosci.com/productivity-uv-vis-spectrophotometer-s-2150uv.html)

Principle of PCR
 Overview
• Polymerase Chain Reaction is a laboratory technique used for the production of a large number of copies DNA
segment
• Developed by Kary Mullis in the 1980s
• Principle – Based on the ability of DNA polymerase enzyme to synthesize a new strand of DNA complementary to
the offered template strand
 A nucleotide can only be added to a pre-existing 3’-OH group, thus a primer to which it can attach is needed –
permits the delineation of a specific sequence to be amplified
 Components of PCR
• DNA template : Sample DNA with target sequence : Exposed to high temperature to separate strands
• Thermostable DNA Polymerase enzyme : Can be obtained from Thermus aquaticus (Taq DNA Polymerase) or
Pyrococcus furiosus (Pfu DNA Polymerase)
Heat resistant enzymes, and can generate new strands using primers
• Primer : short single stranded nucleotide sequence, required for initiating the reaction
- new DNA nucleotides are synthesized from ends of primers

• Deoxy-nucleotidetriphosphates : Used for the synthesis of the new DNA strand
• Buffer solution : maintains chemical environment for reaction process
• Bivalent/Monovalent Cations : Either Mg2+ , Mn2+ , K+ or could be used
 The reaction is carried out in Small Reaction Tubes (0.2-0.5 mL) in a Thermal Cycler (used to maintain high
temperatures)
 Process
• Denaturation
Reaction is heated to 94-98 °C for 20-30 seconds – causes denaturation of DNA (separation of DNA strands due to
breakage of Hydrogen bonds between strands)
• Annealing
Reaction chamber is cooled to 50-65 °C for 20-40 seconds, to allow the attachment of primer to the separated DNA
strands
• Extension/Elongation
Temperature is maintained at 72-75 °C - A new DNA strand complementary to template strand is created by adding
free dNTPs in the 5'-to-3' direction

Diagrammatic Representation of PCR Cycling (https://en.wikipedia.org/wiki/Polymerase_chain_reaction)

 The first 3 steps (Denaturation, Annealing, Extension) are considered as 1 cycle of the reaction – Number of
copies of DNA produced = 2n (Here n is the number of cycles)
Normally 30-40 cycles are performed
• Final elongation
The reaction temperature is kept at 70-74 °C for 5-15 minutes, it ensures that any remaining strands of ssDNA are
fully elongated
• Final Hold
Reaction temperature is reduced to 4-15 °C. The obtained DNA samples may be stored at 4 °C for short periods,
stored at -20 °C or - 80 °C for long term storage
 Stages of PCR replication (PCR Amplification curve)
1. Exponential amplification - At every cycle, the amount of product is doubled (assuming 100% reaction efficiency)
2. Levelling off - The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as
dNTPs and primers, causes them to become more limited
3. Plateau - No more product accumulates due to exhaustion of reagents and enzyme

 Visualization
• Agarose based Gel Electrophoresis – separates DNA fragments based on size (2% Agarose Gel is used)
• Fluorescent based
 Role of House Keeping Genes / Internal Control
• They act as an Internal Control : Increase shows that PCR has been carried out normally
• Positive IC result indicates that amplification has occurred and thus provides assurance that negative test results
are truly negative
• Increases sensitivity by identifying samples inhibitory to PCR
Examples : Beta-Globin, Beta-Actin
https://www.altona-diagnostics.com/en/kit-components-of-realstar-pcr-kits-ruo-ce.html

https://iastate.pressbooks.pub/genagbiotech/chapter/pcr-and-gel-electrophoresis/

Variations of PCR
• Since the technique was invented in the 1980s, PCR has been adapted and modified in many ways, giving rise
to many variations
• To date, there are 37 different variants of PCR used for both diagnostic and research purposes
1. Amplified fragment length polymorphism (AFLP)
PCR
2. Allele-specific PCR
3. Alu PCR
4. Assembly PCR
5. Asymmetric PCR
6. COLD PCR
7. Colony PCR
8. Conventional PCR
9. Digital PCR (dPCR)
10. Fast-cycling PCR
11. High-fidelity PCR
12. High-Resolution Melt (HRM) PCR
13. Hot-start PCR
14. In situ PCR
15. Intersequence-specific (ISSR) PCR
16. Inverse PCR
17. LATE (linear after the exponential) PCR
18. Ligation-mediated PCR
19. Long-range PCR
20. Methylation-specific PCR (MSP)
21. Miniprimer PCR
22.Multiplex-PCR
23.Nanoparticle-Assisted PCR (nanoPCR)
24.Nested PCR
25.Overlap extension PCR
26.Real-Time PCR (quantitative PCR or qPCR)
27.Repetitive sequence-based PCR
28.Reverse-Transcriptase (RT-PCR)
29.Reverse-Transcriptase Real-Time PCR (RT-qPCR)
30.RNase H-dependent PCR (rhPCR)
31.Single cell PCR
32.Single Specific Primer-PCR (SSP-PCR)
33.Solid phase PCR
34.Suicide PCR
35.Thermal asymmetric interlaced PCR (TAIL-PCR)
36.Touch down (TD) PCR
37.Variable Number of Tandem Repeats (VNTR) PCR

Other Variations of PCR
Multiplex PCR
• Involves amplifying multiple sequences in a single reaction – by using several pairs of primers annealing to
different target sequences
• Very useful for DNA fingerprinting, study of genetic mutations, microsatellites and SNPs
Hot Start PCR
• Optimizes the yield of amplified product and suppresses nonspecific amplification and formation of primer dimers
• Done by – inactivating/inhibiting Taq Polymerase and late addition
• Increases product yield
• Provides higher specificity and sensitivity
Reverse Transcriptase PCR (RT-PCR)
• Sample RNA is converted to cDNA (complementary DNA), which is then subjected to PCR cycling and detection
• Useful for detecting RNA viruses in a given sample
• More efficient than Northern Blot method (used for RNA quantification)

Diagram of Hot Start PCR
(https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/genomics/pcr/hot-start-pcr)

Real Time PCR
 It is a laboratory technique used to monitor the amplification of DNA during the reaction, and not at the end
 This is done by 2 methods
1. Adding non specific fluorescent dyes that bind to dsDNA
2. Sequence specific DNA probe with fluorescent reporter that is detected only after hybridization (FRET)
 Non Specific Fluorescent Dyes
• A DNA binding dye is added – binds to dsDNA, therefore increase in dsDNA (product) amount will cause
increased fluorescence intensity
 Fluorogenic Resonance Energy Transfer (Fluorescent reporter probe)
• PCR is prepared, and reporter probe is added - both probe and primers anneal to the DNA target
• Polymerization begins, and once polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe,
separating the fluorescent reporter
• Fluorescence is detected and measured in a real-time PCR machine

Example of Fluorescent probe - 5′-6-carboxyfluorescein-ACGTGGCACTGCGGCACGTGGT-6
(Used to detect Cytomegalovirus DNA)
https://www.thermofisher.com/in/en/home.html

Nested PCR
• Used to increase the specificity of DNA amplification - Two sets of primers are used in two successive reactions
• One pair of primers generates DNA products
• Products from 1st PCR are used as template in 2nd PCR – using two different primers whose binding sites are
located (nested) within the 1st set
• Minimizes non-specific products
Semi-Nested PCR
• Similar to Nested PCR, but 1 primer is common to forward and reverse steps
Asymmetric PCR
• Asymmetric PCR preferentially amplifies one strand of the target DNA - amplified linearly and no longer
exponentially
• Used in some sequencing methods and hybridization probing, to generate one DNA strand as product
• Thermocycling done with a limiting amount of primers
Quantitative PCR
• Used to detect the number of copies of DNA / Pathogen Load
• Two types 1. Absolute Quantitative PCR – Provides absolute measurement of starting copy number
2. Relative Quantitative PCR – Provides accurate discrimination between relative amounts of copy number

Isothermal (Loop Mediated Isothermal Amplification)
• Single-tube technique for the amplification of DNA
• Target sequence is amplified at constant temperature (60–65 °C / 140-149 °F) using two or three sets
of primers and a polymerase with high strand displacement activity
• The amplification product can be detected by photometry or measuring the turbidity caused by
magnesium pyrophosphate precipitate in solution (byproduct of amplification)
• The reaction can be followed in real-time by measuring the turbidity or by fluorescence using intercalating dyes
such as SYTO 9
• Advantages – simple, rugged, and low cost
• Limitations - not useful for cloning
https://international.neb.com/nebinspired-blog/getting-started-with-loop-mediated-isothermal-amplification

References
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC124832/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC87935/
• https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/genomics/pcr/hot-start-pcr
• https://www.frontiersin.org/articles/10.3389/fbioe.2022.1045154/full
• https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/
• https://www.ncbi.nlm.nih.gov/probe/docs/distrperlegen/
• https://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/central_dogma.html
• https://pubmed.ncbi.nlm.nih.gov/29717051/

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