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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

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chapter 7 nucleic acid amplification techniques

Chapter 7: Nucleic Acid Amplification Techniques

Donna C. Sullivan, PhD

Division of Infectious Diseases

University of Mississippi Medical Center

molecular amplification techniques
MOLECULAR AMPLIFICATION TECHNIQUES
  • Nucleic acid (NA) amplification methods fall into 3 categories
    • Target amplification systems
    • Probe amplification systems
    • Signal amplification
target amplification 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
signal and probe amplification methods
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
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
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
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
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
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

AMPLIFICATION BY PCR
polymerase chain reaction
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
Features of Primers
  • Types of primers
    • Random
    • Specific
  • Primer length
    • Annealing temperature
    • Specificity
  • Nucleotide composition
pcr primers
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.
slide15
Tm
  • For short (14–20 bp) oligomers:
    • Tm = 4° (GC) + 2° (AT)
assumptions
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
performing pcr
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
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
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
thermostable polymerases24
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
amplification reaction
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
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
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
Primer Design
  • http://biotools.umassmed.edu/bioapps/primer3_www.cgi
  • 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
product cleanup
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
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
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.
contamination control32

Pre-PCR Post-PCR

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)
polymerase chain reaction controls for pcr
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
interpretation of the pcr results
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.
diagnostic pcr amplification from patient samples

Molecular Marker

Negative Control

Positive Control

Blank Reaction

Patient 1

Patient 2

Patient 3

Patient 4

104 bp

Diagnostic PCR AmplificationFrom Patient Samples
diagnostic pcr amplification from patient samples36

EBV

b-Actin

DNA Marker

Specimen 1

Specimen 1

Specimen 2

Specimen 2

Specimen 1

Specimen 2

Negative

Negative

Positive

Positive

Blank

Diagnostic PCR AmplificationFrom Patient Samples
pcr applications
PCR Applications
  • Structural analysis
  • DNA typing
  • Disease detection
  • Cloning
  • Mutation analysis
  • Detection of gene expression
  • Mapping
  • Site-directed mutagenesis
  • Sequencing
pcr modifications
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
automated pcr and detection
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
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
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
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
qpcr detection systems
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.
slide44

Sample

Threshold

Baseline

No template

quantitative pcr qpcr46

Threshold fluorescence level

Threshold cycles for each sample

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).
qpcr detection systems47
qPCR Detection Systems
  • DNA-specific dyes
    • Ethidium bromide
    • SyBr green
  • Hybridization probes
    • Cleavage-based (TaqMan)
    • Displaceable (Molecular Beacons, FRET)
  • Primer-incorporated probes
dna detection sybr green i dye
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

slide49

qPCR: SyBr Green

  • Binds minor groove of double-

stranded DNA.

  • Product can be further tested

in a post-amplification melt

curvein which sequences

have characteristic melting

temperatures.

real time pcr labeled probes
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
cleavage based assay taqman 5 3 exonuclease
Cleavage-based Assay: TaqMan 5’-3’ Exonuclease

Dual labeled Probe

Cleavage of Dual labeled Probe

hybridization probe format for dna detection
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 systems55
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

real-time

real-time

real-time PCR

hardware

5700

Applied Biosystems

iCycler

BioRad

7700

Applied Biosystems

LightCycler

Roche

FluorTracker

Stratagene

FluorImager

Molecular Dynamics

real-time

Real Time PCR Instrumentation
pcr advantages
PCR Advantages
  • Specific
  • Simple, rapid, relatively inexpensive
  • Amplifies from low quantities
  • Works on damaged DNA
  • Sensitive
  • Flexible
pcr limitations
PCR Limitations
  • Contamination risk
  • Primer complexities
  • Primer-binding site complexities
  • Amplifies rare species
  • Detection methods
target amplification methods59
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
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
probe and signal amplification methods
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
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
slide65

Ligase Chain Reaction

Template

Probes

...GTACTCTAGCT...

A G

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 Amplification of Genomic DNA
ligase chain reaction mutation detection utilizing mutant specific oligonucleotide primers

Wild-Type Sequence

Mutant Sequence

Annealing

Ligation

No DNA Products

DNA Product

Ligase Chain Reaction Mutation Detection: Utilizing Mutant-Specific Oligonucleotide Primers
branched dna detection
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
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 detection71

Hybridize

bDNA Probe

Target: Capture Probe

Hybrid

Hybridize

bDNA Amplifier

Addition of

Alkaline Phosphatase

Molecules

Branched DNA Detection
hybrid capture assay
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 assay74
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 assay75
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
Web Sites of Interest
  • http://www.genscript.com/custom_service.html?&gs_cust=391826&gs_camp=316
  • http://www.bio.davidson.edu/courses/genomics/chip/chip.html
summary
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.