PCR and RT PCR

Dr. Murali Mohan Challa

Professor
Department of Biotechnology
Visakhapatnam - 530045
https://www.youtube.com/watch?v=fl3_ttDa89g Email : [email protected]
https://www.youtube.com/watch?v=-m_Xa4Gfu8A
2 September 2024
www.gitam.edu
BTEN40712: Molecular Diagnostics and its applications 1
 Learning Outcomes
• After completion this topic, the student will be able to
– explain PCR Technique(L2).
– applications of PCR Technique (L2).
– explain RT PCR technique (L2).
– explain application of RT PCR (L2).

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 Objective of today’s Class
• What is PCR?
– PCR Technique
– Applications of PCR Technique.
– What is RT PCR and how it is performed?
– Applications of RT-PCR.

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 Historical background
• The Polymerase Chain Reaction (PCR) technique, invented in 1985 by Kary B. Mullis, allowed scientists to make
millions of copies of a scarce sample of DNA.
• The technique is also used by criminologists to link specific persons to samples of blood or hair via DNA
comparison.
• Polymerase chain reaction (PCR) is a laboratory technique used to make multiple copies of a segment of
DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA
molecules.
• A PCR reaction consists of three steps: denaturation, annealing, and extension.

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Kary B. Mullis and the Nobel Prize: The
Basics
• Developed the process in 1986 and won Nobel prize in 1993.
• Knew that you could expose template DNA by boiling ds DNA to
produce ss DNA
• Knew that you could use primers to initiate DNA synthesis
• Knew that a cheap, commercial enzyme was available (Klenow
fragment of E. coli DNA polymerase)

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 Cary Mullis and PCR

• Wanted a way to generate large amounts of DNA

from a single copy
• Initially used the “3 graduate student” method
– Denaturing
– Annealing
– Extending

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 THREE STEPS OF PCR

• Denaturation of target (template)

– Usually 95oC
• Annealing of primers
– Temperature of annealing is dependent on the G+C content
– May be high (no mismatch allowed) or low (allows some mismatch)
stringency
• Extension (synthesis) of new strand

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 AMPLIFICATION BY PCR

5’
Target 3’
3’
5’
1. Denature
2. Anneal primers

3. Extend primers
Two copies
of target
1. Denature

2. Anneal primers
3. Extend primers
Four copies
of target

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 PCR: First 4 Cycles

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 PCR: Completed Amplification
Cycle

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 POLYMERASE CHAIN REACTION

• Primers (may be specific or random)

• Thermostable polymerase
– Taq pol
– Pfu pol
– Vent pol
• Target nucleic acid (template)
– Usually DNA
– Can be RNA if an extra step is added

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 Features of Primers

• Types of primers
– Random
– Specific
• Primer length
– Annealing temperature
– Specificity
• Nucleotide composition

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 PCR Primers

• Primers are single-stranded 18–30 b DNA fragments complementary to

sequences flanking the region to be amplified.

• Primers determine the specificity of the PCR reaction.

• The distance between the primer binding sites will determine the size
of the PCR product.

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 PCR
Tm:
• For short (14–20 bp) oligomers:
– Tm = 4° (GC) + 2° (AT)
Assumptions:
• Product produced is product desired
– There is always the possibility of mismatch and production of artifacts
– However, if it is the right size, its probably the right product
• Product is from the orthologous locus
– Multigene families and pseudogenes

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 Thermostable DNA Polymerase:
Yellowstone National Park

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 Thermostable Polymerases
Polymerase T ½, Extension Type of Source
95oC Rate (nt/sec) ends
Taq pol 40 min 75 3’A T. aquaticus

Amplitaq 80 min >50 3’A T. aquaticus

(Stoffel
fragment)
Vent* 400 min >80 95%
Thermococcus
blunt
litoralis
Deep Vent* 1380 min ? 95%
Pyrococcus
blunt
GB-D
Pfu >120 min 60 Blunt
Pyrococcus
furiosus
Tth* 20 min >33 3’A T.
(RT activity) thermophilus
*Have proof-reading functions and can generate products over
30 kbp

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 Performing PCR

• Assemble a reaction mix containing all components necessary for DNA

synthesis.
• Subject the reaction mix to an amplification program.
• Analyze the product of the PCR reaction (the amplicon).

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 A Standard PCR Reaction Mix

0.25 mM each primer

0.2 mM each dATP, dCTP, dGTP, dTTP
50 mM KCl
10 mM Tris, pH 8.4
1.5 mM MgCl2
2.5 units polymerase
102 - 105 copies of template
50 ml reaction volume

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 PCR Cycle: Temperatures
• Denaturation temperature
– Reduce double stranded molecules to single stranded molecules
– 90–96oC, 20 seconds
• Annealing temperature
– Controls specificity of hybridization
– 40–68oC, 20 seconds
• Extension temperature
– Optimized for individual polymerases
– 70–75oC, 30 seconds

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 Combinations Of Cycle
Temperatures
TEMP FOR COMMENTS
94-60-72 Perfect, long Higher temp can be used;
primers maximum annealling temp
94-55-72 Good or perfectly Standard conditions
matched primers
between 19-24 nt
94-50-72 Adequate primers Allows 1-3 mismatches/20 nt

94-48-68 Poorly matched Allows 4-5 mismatches/20 nt

primers
94-45-65 Unknown match, Primers of questionable
likely poor quality, long-shot PCR
94-37-65 Hail Mary Uncontrolled results

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 Thermostable Polymerases

• Taq: Thermus aquaticus (most commonly used)

– Sequenase: T. aquaticus YT-1
– Restorase (Taq + repair enzyme)
• Tfl: T. flavus
• Tth: T. thermophilus HB-8
• Tli: Thermococcus litoralis
• Carboysothermus hydrenoformans (RT-PCR)
• P. kodakaraensis (Thermococcus) (rapid synthesis)
• Pfu: Pyrococcus furiosus (fidelity)
– Fused to DNA binding protein for processivity

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 Amplification Reaction

• Amplification takes place as the reaction mix is subjected to an

amplification program.

• The amplification program consists of a series of 20–50 PCR cycles.

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 Automation of PCR
• PCR requires repeated temperature changes.

• The thermal cycler changes temperatures in a block or chamber holding

the samples.

• Thermostable polymerases are used to withstand the repeated high

denaturation temperatures.

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 Avoiding Misprimes
• Use proper annealing temperature.
• Design primers carefully.
• Adjust monovalent cation concentration.
• Use hot-start: prepare reaction mixes on ice, place in preheated cycler or use a sequestered
enzyme that requires an initial heat activation.
– Platinum Taq
– AmpliTaq Gold
– HotStarTaq

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 Primer Design

• http://biotools.umassmed.edu/bioapps/primer3_www.cg
i
• http://arbl.cvmbs.colostate.edu/molkit/rtranslate/index.
html
• Avoid inter-strand homologies
• Avoid intra-strand homologies
• Tm of forward primer = Tm of reverse primer
• G/C content of 20–80%; avoid longer than GGGG
• Product size (100–700 bp)
• Target specificity

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 Product Cleanup

• Gel elution
– Removes all reaction components as well as misprimes and primer dimers
• Solid phase isolation of PCR product (e.g., spin columns)
• DNA precipitation

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 Contamination Control
• Any molecule of DNA containing the intended target sequence is a
potential source of contamination.
• The most dangerous contaminant is PCR product from a previous reaction.
• Laboratories are designed to prevent exposure of pre-PCR reagents and
materials to post-PCR contaminants.

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Contamination of PCR Reactions
• Most common cause is carelessness and bad technique.
• Separate pre- and post-PCR facilities.
• Dedicated pipettes and reagents.
• Change gloves.
• Aerosol barrier pipette tips.
• Meticulous technique
• 10% bleach, acid baths, UV light
• Dilute extracted DNA.

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 Contamination Control
Pre-PCR Post-PCR
• Physical separation
– Air-locks, positive air flow
– PCR hoods with UV
• dUTP + uracil-N-glycosylase (added to the PCR reaction)
• Psoralen + UV (depends on UV wavelength and distance to
surface)
• 10% bleach (most effective for surface decontamination)

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 Polymerase Chain Reaction
Controls for PCR
• Blank reaction
– Controls for contamination
– Contains all reagents except DNA template
• Negative control reaction
– Controls for specificity of the amplification reaction
– Contains all reagents and a DNA template lacking the target sequence
• Positive control reaction
– Controls for sensitivity
– Contains all reagents and a known target-containing DNA template

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 Interpretation of the PCR Results

• The PCR product should be of the expected size.

• No product should be present in the reagent blank.
• Misprimes may occur due to non-specific hybridization of primers.
• Primer dimers may occur due to hybridization of primers to each
other.

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 Diagnostic PCR Amplification
From Patient Samples

104 bp

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 Diagnostic PCR Amplification
From Patient Samples
EBV b-Actin

DNA Marker
Specimen 1

Specimen 1

Specimen 2

Specimen 2

Specimen 1

Specimen 2
Negative

Negative
Positive

Positive
Blank

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 PCR Applications

• Structural analysis
• DNA typing
• Disease detection
• Cloning
• Mutation analysis
• Detection of gene expression
• Mapping
• Site-directed mutagenesis
• Sequencing

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 PCR Modifications
• PCR- RFLP
• Real time PCR
• Reverse-transcriptase PCR
• Multiplex PCR

https://www.youtube.com/watch?v=fl3_ttDa89g

2 September 2024 BTEN40712: Molecular Diagnostics and its applications 35

 PCR - RFLP

https://www.youtube.com/watch?v=n2bVFcJP-HY
2 September 2024 BTEN40712: Molecular Diagnostics and its applications 36
 PCR - RFLP
• PCR has greatly improved the sensitivity of detecting mutations in
genomic DNA
• The general Scheme of PCR-based RFLP includes amplification of DNA
containing the mutated sequence using flanking primers, followed by
enzyme restriction of the PCR product.
• K-ras codon 12 mutation have been detected in gastric carcinoma by
simple gel electrophoresis of the PCR product cleaved with the
restriction endonuclease HPA II (5’CCGG) – (Cut results 5’ C CGG.)

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 PCR - RFLP
• Because any substitution of the first two nucleotides of codon 12
abolishes the restriction site of this enzyme, undigested product
revealed by simple gel electrophoresis indicates a mutation.
• As few as 10 copies of mutated k-ras gene in 109 wild type sequences
have been detected using such mutant-enhancement using PCR-RFLP
technology.
• Transforming any alteration in DNA sequence into an allele –specific
enzyme recognition site obviates the use of radioisotopic hybridization
and has been successfully to detect multiple mutations in the cystic
fibrosis genes as well as ras oncogenes in gastrointestinal cancers
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 PCR - RFLP
• In addition, primer-mediated restriction polymorphism has enhanced
by 20% the sensitivity of detection of deletion of codon 12 k-ras
mutation in colorectal cancers, as compared to allele – specific
oligonucleotide hybridisation technique.

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 Reverse Transcriptase-PCR

• RT–PCR is a variation of PCR, or polymerase chain

reaction.

• The two techniques use the same process except that RT–
PCR has an added step of reverse transcription of RNA to
DNA, or RT, to allow for amplification.

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 Reverse transcriptase PCR
• Reverse transcriptase-PCR is to amplify cDNA copies of RNA
• It is used to retrieve and clone the 5’ and 3’ termini of mRNA
• It is used to generate large cDNA libraries from very small amounts of
mRNA
• It can be adapted to identify mutations and polymorphisms
• It can be used to measure the strength of gene expression

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 Convertion of mRNA into cDNA

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• Mesophilic enzymes encoded by avian myeloblastosis virus (AMV) or
Moloney strain of murine leukaemia virus (Mo-MLV).
• Variants of Mo-MLV reverse transcriptase that lacks RNase H activity.
• Thermostable Tth DNA polymerase (exhibit RT activity in presence of
Mn2+)

• Three ways to amplify cDNA

• 1. Gen specific priming 2. Oligo dT priming 3. Random hexamer
priming.

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 Action of three primers

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 RT PCR for Gene expression

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 Reverse Transcriptase-PCR

https://www.youtube.com/watch?v=1vqNZ-H7Pq0

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 Real-Time or Quantitative PCR
(qPCR)
• Standard PCR with an added probe or dye to generate a fluorescent
signal from the product.
• Detection of signal in real time allows quantification of starting material.
• Performed in specialized thermal cyclers with fluorescent detection
systems.
https://www.youtube.com/watch?v=Dho2ddDmam8

https://www.youtube.com/watch?v=iu4s3Hbc_bw https://www.youtube.com/watch?v=_pMCwREQ22I

https://www.youtube.com/watch?v=LBrVspSqDj4 https://www.youtube.com/watch?v=tH_ozcFwQ_Q
https://www.youtube.com/watch?v=XH6vIBLwC2M

2 September 2024 BTEN40712: Molecular Diagnostics and its applications 49

 Quantitative PCR (qPCR)

• PCR product grows in an exponential fashion (doubling at each cycle).

• PCR signal is observed as an exponential curve with a lag phase, a log
phase, a linear phase, and a stationary phase.
• The length of the lag phase is inversely proportional to the amount of
starting material.

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 Real-Time PCR Labeled Probes
• Cleavage-based probes
– TaqMan Assay
– Fluorescent reporter at 5’ end and a quencher at 3’ end
• Molecular beacons
– Hairpin loop structure
– Fluorescent reporter at 5’ end and a quencher at 3’ end
• FRET probes
– Fluorescence resonance energy transfer probes

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 Real-Time PCR

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 Real-Time PCR

• TaqMan probe method

• SYBR green method

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 Real Time PCR Instrumentation

5700
Applied Biosystems

iCycler
BioRad

7700
Applied Biosystems

LightCycler
real-time
Roche

real-time
real-time PCR FluorTracker
real-timehardware Stratagene

FluorImager
Molecular Dynamics

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 PCR Advantages

Specific

Simple, rapid, relatively inexpensive

Amplifies from low quantities

Works on damaged DNA

Sensitive

Flexible

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 PCR Limitations

• Contamination risk
• Primer complexities
• Primer-binding site complexities
• Amplifies rare species
• Detection methods

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