Chapter 7: Nucleic Acid
Amplification
Techniques
Donna C. Sullivan, PhD
Division of Infectious Diseases
University of Mississippi Medical
Center
MOLECULAR AMPLIFICATION
TECHNIQUES

Nucleic acid (NA) amplification methods fall
into 3 categories



Target amplification systems
Probe amplification systems
Signal amplification
Target Amplification Methods

PCR –



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
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PCR using specific probes
RT PCR
Nested PCR-increases sensitivity, uses two sets of
amplification primers, one internal to the other
Multiplex PCR-two or more sets of primers specific for
different targets
Arbitrarily Primed PCR/Random Primer PCR
NASBA - Nucleic Acid Sequence-Based
Amplification
TMA – Transcription Mediated Amplification
SDA - Strand Displacement Amplification
Signal and Probe Amplification
Methods

Signal Amplification



bDNA – Branched DNA probes
Hybrid Capture – Anti-DNA-RNA hybrid antibody
Probe Amplification


LCR – Ligase Chain Reaction
Cleavase Invader – FEN-1 DNA polymerase
(cleavase)
TARGET AMPLIFICATION
TECHNIQUES




All use enzyme-mediated processes, to
synthesize copies of target nucleic acid
Amplification products detected by 2
oligonucleotide primers
Produce 108-109 copies of targeted
sequences
Sensitive to contamination, false-positive
reaction
Kary Mullis and the Nobel
Prize: The Basics



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)
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
THREE STEPS OF PCR

Denaturation of target (template)


Annealing of primers



Usually 95oC
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
AMPLIFICATION BY PCR
Target
5’
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
PCR: First 4 Cycles
PCR: Completed Amplification
Cycle
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
Features of Primers

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
Types of primers
 Random
 Specific
Primer length
 Annealing temperature
 Specificity
Nucleotide composition
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.

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
Thermostable DNA Polymerase:
Yellowstone National Park
Alvin Submersible for
Exploration of Deep Sea Vents
Thermostable Polymerases
Polymerase
Taq pol
Amplitaq
(Stoffel
fragment)
Vent*
T ½,
95oC
40 min
Extension Type of
Rate (nt/sec)
ends
75
3’A
80 min
>50
3’A
400 min
>80
95%
blunt
95%
blunt
Blunt
Source
T. aquaticus
T. aquaticus
Thermococcus
litoralis
Deep Vent* 1380 min
?
Pyrococcus
GB-D
Pfu
>120 min
60
Pyrococcus
furiosus
Tth*
20 min
>33
3’A
T.
(RT activity)
thermophilus
*Have proof-reading functions and can generate products over
30 kbp
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).

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
PCR Cycle: Temperatures

Denaturation temperature



Annealing temperature



Reduce double stranded molecules to single stranded
molecules
90–96oC, 20 seconds
Controls specificity of hybridization
40–68oC, 20 seconds
Extension temperature


Optimized for individual polymerases
70–75oC, 30 seconds
Combinations Of Cycle
Temperatures
TEMP
94-60-72
94-55-72
94-50-72
94-48-68
94-45-65
94-37-65
FOR
COMMENTS
Perfect, long
primers
Good or perfectly
matched primers
between 19-24 nt
Adequate primers
Higher temp can be used;
maximum annealling temp
Standard conditions
Poorly matched
primers
Unknown match,
likely poor
Hail Mary
Allows 4-5 mismatches/20 nt
Allows 1-3 mismatches/20 nt
Primers of questionable
quality, long-shot PCR
Uncontrolled results
Thermostable Polymerases


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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
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.
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.
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
Primer Design


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http://biotools.umassmed.edu/bioapps/primer3_www.
cgi
http://arbl.cvmbs.colostate.edu/molkit/rtranslate/inde
x.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
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
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.
Contamination of PCR
Reactions
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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.
Contamination Control

Physical separation

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Air-locks, positive air flow
PCR hoods with UV
dUTP + uracil-Nglycosylase (added to the
PCR reaction)
Psoralen + UV (depends on
UV wavelength and
distance to surface)
10% bleach (most effective
for surface
decontamination)
Pre-PCR Post-PCR
Polymerase Chain Reaction
Controls for PCR

Blank reaction

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Negative control reaction

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Controls for contamination
Contains all reagents except DNA template
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
Interpretation of the PCR
Results


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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.
Diagnostic PCR Amplification
From Patient Samples
104 bp
Specimen 2
Specimen 1
Positive
EBV
Negative
DNA Marker
Specimen 2
Specimen 2
Specimen 1
Specimen 1
Positive
Negative
Blank
Diagnostic PCR Amplification
From Patient Samples
b-Actin
PCR Applications
Structural analysis
 DNA typing
 Disease detection
 Cloning
 Mutation analysis
 Detection of gene expression
 Mapping
 Site-directed mutagenesis
 Sequencing

PCR Modifications
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Nested PCR
Multiplex PCR
Tailed primers
Sequence-specific PCR
Reverse-transcriptase PCR
Long-range PCR
Whole-genome amplification
RAPD PCR (AP-PCR)
Quantitative real-time PCR
Automated PCR and Detection

The COBAS Amplicor Analyzer



Samples are amplified and products detected
automatically after the PCR reaction
Used for infectious disease applications (HIV,
HCV, HBV, CMV, Chlamydia, Neisseria,
Mycobacterium tuberculosis)
Real-time or quantitative PCR (qPCR)

Products are detected by fluorescence during the
PCR reaction
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.
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.
SEQUENCE DETECTION
APPLICATIONS

End point PCR: simple +/- results

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Real time PCR: complex results

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PCR product detection (pathogens, transgenes)
Genotyping (allelic discrimination, single
nucleotide polymorphisms-SNPs)
Absolute quantitation
Relative quantitation
PCR interrogation (optimization)
Hybridization analysis: probe hybridization
qPCR Detection Systems



DNA-specific dyes bind and fluoresce doublestranded DNA nonspecifically.
Hybridization probes only bind and fluoresce
the intended PCR product.
Primer-incorporated probes label the PCR
product.
MODEL OF SINGLE AMPLIFICATION
PLOT
1
0.9
0.8
Sample
0.7
0.5
0.4
Threshold
0.3
0.2
Baseline
0.1
Ct
41
37
33
29
21
17
13
9
5
0
25
No template
1
Rn
0.6
GEL ANALYSIS VS
FLUORESCENCE
Quantitative PCR (qPCR)


A threshold level of
fluorescence is
determined based on
signal and
background.
Input is inversely
proportional to
“threshold” cycle
(cycle at which
fluorescence crosses
the threshold
fluorescence level).
Threshold
fluorescence level
Threshold cycles for each sample
qPCR Detection Systems

DNA-specific dyes

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
Hybridization probes

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
Ethidium bromide
SyBr green
Cleavage-based (TaqMan)
Displaceable (Molecular Beacons, FRET)
Primer-incorporated probes
DNA Detection: SYBR Green I
Dye
DENATURATION STEP: DNA + PRIMERS + DYE
WEAK BACKGROUND FLUORESCENCE
ANEALING STEP:DYE
BINDS dsDNA, EMITS LIGHT
EXTENSION STEP: MEASURE
LIGHT EMMISSION
qPCR: SyBr Green
 Binds minor groove of doublestranded DNA.
 Product can be further tested
in a post-amplification melt
curve in which sequences
have characteristic melting
temperatures.
Real-Time PCR Labeled
Probes

Cleavage-based probes

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Molecular beacons

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
TaqMan Assay
Fluorescent reporter at 5’ end and a quencher at 3’ end
Hairpin loop structure
Fluorescent reporter at 5’ end and a quencher at 3’ end
FRET probes

Fluorescence resonance energy transfer probes
Cleavage-based Assay: TaqMan 5’-3’
Exonuclease
Dual labeled Probe
Cleavage of Dual labeled Probe
Molecular Beacon Assay
FRET Probe
HYBRIDIZATION PROBE FORMAT FOR DNA
DETECTION
DENATURATION STEP: DNA +
TWO FLUORESCENT PROBES
ANNEALING STEP: PROBES BIND
VERY NEAR ONE ANOTHER
EXTENSION STEP: ENERGY OF EXCITATION
FROM ONE PROBE TRANSFERRED TO THE
OTHER (FLUORESECENCE RESONANCE
ENERGY TRANSFER, FRET)
qPCR Detection Systems
Thermal cyclers with fluorescent detection
and specialized software.
 PCR reaction takes place in optically clear
plates, tubes, or capillaries.

Cepheid
Smart Cycler
Roche
LightCycler
Real Time PCR
Instrumentation
5700
Applied Biosystems
iCycler
BioRad
7700
Applied Biosystems
LightCycler
real-time
real-time
real-time PCR
Roche
real-timehardware
FluorTracker
Stratagene
FluorImager
Molecular Dynamics
PCR Advantages
Specific
 Simple, rapid, relatively inexpensive
 Amplifies from low quantities
 Works on damaged DNA
 Sensitive
 Flexible

PCR Limitations
Contamination risk
 Primer complexities
 Primer-binding site complexities
 Amplifies rare species
 Detection methods

Target Amplification Methods

PCR –








PCR using specific probes
RT PCR
Nested PCR-increases sensitivity, uses two sets of amplification
primers, one internal to the other
Multiplex PCR-two or more sets of primers specific for different
targets
Arbitrarily Primed PCR/Random Primer PCR
NASBA - Nucleic Acid Sequence-Based
Amplification
TMA – Transcription Mediated Amplification
SDA - Strand Displacement Amplification
TRANSCRIPTION AMPLIFICATION
METHODS




Nucleic acid sequence based amplification (NASBA)
and transcription mediated amplification (TMA)
Both are isothermal RNA amplifications modeled
after retroviral replication
RNA target is reverse transcribed into cDNA,
followed by RNA synthesis via RNA polymerase
Amplification involves synthesis of cDNA from RNA
target with a primer containing the T7 RNA pol
promoter sequence
Both NASBA and TMA Begin
with RNA
Probe and Signal Amplification
Methods

Probe Amplification




LCR – Ligase Chain Reaction
Strand Displacement Amplification
Cleavase Invader – FEN-1 DNA polymerase
(cleavase)
Signal Amplification


bDNA – Branched DNA probes
Hybrid Capture – Anti-DNA-RNA hybrid antibody
Ligase Chain Reaction
Isothermal
 Probe amplification
 Probes bind immediately adjacent to one another
on template.
 The bound probes are ligated and become
templates for the binding of more probes.
 C. trachomatis, N. gonorrhoeae, sickle cell
mutation

Ligase Chain Reaction
Template
Probes
...GTACTCTAGCT...
AG
T C
...CATGAGATCGA...
ligase
Target sequences are detected by coupled
and
.
Ligase Chain Reaction
Amplification of Genomic DNA
Primer 1
Target
Primer 2
Target
Annealing
Ligation
Additional cycles of
denaturation, annealing, ligation
Ligase Chain Reaction Mutation
Detection: Utilizing Mutant-Specific
Oligonucleotide Primers
Wild-Type Sequence
Mutant Sequence
Annealing
Ligation
No DNA Products
DNA Product
Strand Displacement
Amplification
Branched DNA Detection




Target nucleic acid sequences are not replicated
through enzymatic amplification.
Detection sensitivity is provided by amplification of
the signal from the probe.
Uses “capture probes,” “bDNA probes” and “bDNA
amplifier probes.”
Assay is based upon microtiter plate technology.
bDNA ASSAYS


Solid phase signal amplification system
Multiple sets of synthetic oligonucleotide
probes



Capture probes bound to well
Target specific probes
Amplifier molecule with 15 identical branches,
each of which can bind to 3 labeled probes
Branched DNA Detection
Hybridize
bDNA Probe
Target: Capture Probe
Hybrid
Hybridize
bDNA Amplifier
Addition of
Alkaline Phosphatase
Molecules
bDNA ASSAYS
HYBRID CAPTURE ASSAY


Solution hybridization, antibody capture
assay
Chemiluminescence detection of hybrid
(DNA/RNA) molecules



DNA is denatured
Hybridized to RNA probe
Captured by bound anti DNA/RNA antibodies
Hybrid Capture Assay

Release Nucleic Acids


Clinical specimens are
combined with a base
solution which disrupts
the virus or bacteria and
releases target DNA.
Hybridize RNA Probe
with Target DNA

Target DNA combines
with specific RNA probes
creating RNA:DNA
hybrids.
Hybrid Capture Assay

Capture Hybrids


RNA:DNA hybrids are
captured onto a
microtiter well coated
with capture antibodies
specific for RNA:DNA
hybrids.
Label for Detection

Captured RNA:DNA
hybrids are detected with
multiple antibodies
conjugated to alkaline
phosphatase
Web Sites of Interest


http://www.genscript.com/custom_service.ht
ml?&gs_cust=391826&gs_camp=316
http://www.bio.davidson.edu/courses/genomi
cs/chip/chip.html
Summary





PCR is a method to specifically amplify target
sequences in a complex mixture.
The primers determine what sequences are
amplified (specificity).
Contamination control is important in laboratories
performing PCR.
Quantitative PCR offers the advantage of
quantifying target.
In addition to PCR, signal and probe amplification
methods are available for use in the clinical
laboratory.