Chapter 7: Nucleic Acid Amplification Techniques

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Chapter 7 Nucleic Acid Amplification Techniques

• Donna C. Sullivan, PhD
• Division of Infectious Diseases
• University of Mississippi Medical Center

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MOLECULAR AMPLIFICATION TECHNIQUES

• Nucleic acid (NA) amplification methods fall into
  3 categories
• Target amplification systems
• Probe amplification systems
• Signal amplification

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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

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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)

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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

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Cary 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)

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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 GC
  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
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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 1830 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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Tm

• For short (1420 bp) oligomers
• Tm 4 (GC) 2 (AT)

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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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Alvin Submersible for Exploration of Deep Sea
Vents
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Thermostable Polymerases
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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
• 9096oC, 20 seconds
• Annealing temperature
• Controls specificity of hybridization
• 4068oC, 20 seconds
• Extension temperature
• Optimized for individual polymerases
• 7075oC, 30 seconds

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Combinations Of Cycle Temperatures
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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
  2050 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.c
  gi
• 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 2080 avoid longer than GGGG
• Product size (100700 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

• 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 ReactionControls 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 AmplificationFrom Patient Samples
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Diagnostic PCR AmplificationFrom Patient Samples
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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

• 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

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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

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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.

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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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SEQUENCE DETECTION APPLICATIONS

• End point PCR simple /- results
• PCR product detection (pathogens, transgenes)
• Genotyping (allelic discrimination, single
  nucleotide polymorphisms-SNPs)
• Real time PCR complex results
• Absolute quantitation
• Relative quantitation
• PCR interrogation (optimization)
• Hybridization analysis probe hybridization

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qPCR Detection Systems

• DNA-specific dyes bind and fluoresce
  double-stranded DNA nonspecifically.
• Hybridization probes only bind and fluoresce the
  intended PCR product.
• Primer-incorporated probes label the PCR product.

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Sample
Threshold
Baseline
No template
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GEL ANALYSIS VS FLUORESCENCE
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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).

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qPCR Detection Systems

• DNA-specific dyes
• Ethidium bromide
• SyBr? green
• Hybridization probes
• Cleavage-based (TaqMan?)
• Displaceable (Molecular Beacons?, FRET?)
• Primer-incorporated probes

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DNA Detection SYBR Green I Dye
DENATURATION STEP DNA PRIMERS DYE WEAK
BACKGROUND FLUORESCENCE
ANEALING STEPDYE BINDS dsDNA, EMITS LIGHT
EXTENSION STEP MEASURE LIGHT EMMISSION
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qPCR SyBr? Green

• Binds minor groove of double-
• stranded DNA.
• Product can be further tested
• in a post-amplification melt
• curve in which sequences
• have characteristic melting
• temperatures.

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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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Cleavage-based Assay TaqMan 5-3 Exonuclease
Dual labeled Probe
Cleavage of Dual labeled Probe
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Molecular Beacon Assay
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FRET Probe
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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)
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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
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Real Time PCR Instrumentation
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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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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

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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

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Both NASBA and TMA Begin with RNA
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(No Transcript)
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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

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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

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Ligase Chain Reaction
Template
Probes
...GTACTCTAGCT...
A G
T C
...CATGAGATCGA...
ligase

Target sequences are detected by coupled and
.
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Ligase Chain Reaction Amplification of Genomic
DNA
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Ligase Chain Reaction Mutation Detection
Utilizing Mutant-Specific Oligonucleotide Primers
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Strand Displacement Amplification
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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.

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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

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Branched DNA Detection
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bDNA ASSAYS
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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

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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 RNADNA hybrids.

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Hybrid Capture Assay

• Capture Hybrids
• RNADNA hybrids are captured onto a microtiter
  well coated with capture antibodies specific for
  RNADNA hybrids.
• Label for Detection
• Captured RNADNA hybrids are detected with
  multiple antibodies conjugated to alkaline
  phosphatase

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Web Sites of Interest

• http//www.genscript.com/custom_service.html?gs_c
  ust391826gs_camp316
• http//www.bio.davidson.edu/courses/genomics/chip/
  chip.html

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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.