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Nucleic Acid Amplification

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Title: Nucleic Acid Amplification


1
Nucleic Acid Amplification
  • Donna C. Sullivan, PhD
  • Division of Infectious Diseases
  • University of Mississippi Medical Center

2
Objectives
  • Describe the principle of amplification by
    polymerase chain reaction (PCR).
  • Discuss how products are detected.
  • Describe examples of modifications that have been
    developed for PCR.
  • Differentiate between target amplification and
    signal amplification.
  • Compare and contrast between the following in
    vitro assays for amplifying nucleic acids PCR,
    branched DNA, ligase chain reaction,
    transcription mediated amplification, and hybrid
    capture.

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

4
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

5
Polymerase Chain Reaction
  • Primer-directed in vitro enzymatic reaction for
    the production of a specific DNA fragment.

6
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)

7
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

8
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

9
AMPLIFICATION BY PCR
10
PCR Step 1 Denaturation
11
PCR Step 2 Annealing
12
PCR Step 3 Extension
13
PCR Cycle 2
14
End of PCR Cycle 2
15
  • PCR product amplicon
  • The number of amplicons 2N where N the
  • number of cycles

16
(No Transcript)
17
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18
Components and Results of a PCR
19
Features of Primers
  • Types of primers
  • Random
  • Specific
  • Primer length
  • Annealing temperature
  • Specificity
  • Nucleotide composition

20
DESIGNING PRIMERS
  • Random amplification primers usually short
  • 10-18 nt in length
  • Vary percent G C content
  • Longer the primer, the higher the annealling
    temperature
  • The higher the annealling temperature, the
    greater the specificity

21
DESIGNING PRIMERS
  • Nucleotide composition
  • Self-annealling (primer dimers)
  • High G C content allows higher annealling
    temperature but increases risk of self-annealling
  • Primer-template match
  • Get products even when primer/template not
    perfectly matched
  • Single internal mismatch with 6-10 matches on
    either side will have little effect on PCR
    product yield

22
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

23
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.

24
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).

25
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

26
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.

27
In Vitro Amplification
How much amplification do you get? Amplicons A
X 2n2 n number of cycles A starting target
copy number Reagent limitations and polymerase
infidelity result in plateau effect (reduced
amplification efficiency).
28
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.

29
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

30
Thermostable DNA Polymerase Yellowstone
National Park
31
Alvin Submersible for Exploration of Deep Sea
Vents
32
Thermostable Polymerases
33
PCR Cycle Temperatures
  • Denaturation temperature
  • Reduce double stranded molecules to single
    stranded molecules
  • Annealing temperature
  • Controls specificity of hybridization
  • Extension temperature
  • Optimized for individual polymerases

34
Combinations Of Cycle Temperatures
35
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.

36
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

37
Analysis of PCR Products
38
Diagnostic PCR AmplificationFrom Patient Samples
39
Diagnostic PCR AmplificationFrom Patient Samples
40
Detection of PCR Product
Reagent blank
Molecular weight markers
Misprime
PCR product
Primer dimers
41
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


42
Primer Design
  • 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

43
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

44
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.

45
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)

46
Advantages of PCR
  • PCR is fast (25 hours).
  • DNA or RNA can be amplified.
  • High-yield amplification can be achieved (106 to
    109 amplification).
  • DNA from one cell equivalent can be amplified.
  • PCR products can be directly sequenced.
  • PCR products can be directly cloned.
  • DNA sequences up to 30 kb can be amplified.

47
PCR Advantages
  • Specific
  • Simple, rapid, relatively inexpensive
  • Amplifies from low quantities
  • Works on damaged DNA
  • Sensitive
  • Flexible

48
PCR Limitations
  • Contamination risk
  • Primer complexities
  • Primer-binding site complexities
  • Amplifies rare species
  • Detection methods

49
Disadvantages of PCR
  • Must know the sequence of the DNA of interest.
  • Highly susceptible to contamination or false
    amplification.
  • Amplification may not be 100 specific.
  • Specificity of amplification is dependent on
    temperature and Mg concentration.
  • Analysis and product detection usually takes
    longer than the PCR reaction itself.

50
Troubleshooting PCR
  • No PCR product
  • Verify that all components were added to the
    reaction.
  • Check pipetors and reagents.
  • Check detection method.
  • Too many bands
  • Specificity of primers.
  • Annealing temp too low, excessive Mg or cycles.

51
Troubleshooting PCR (cont.)
  • Primer dimers
  • Size is the sum of two primer lengths.
  • Taq extends one primer, which is annealed to
    another primer.
  • Annealing temperature too low, excess primers.

52
PCR Inhibitors
  • Detergent
  • Phenol
  • Heparin
  • Heme
  • Dyes (bromphenol blue)
  • CSF, urine, sputum, paraffin

Dilute extracted DNA.
53
Contamination of PCR Reactions
  • Most common cause is carelessness and bad
    technique.
  • Separate pre- and post-PCR facilities.
  • Dedicated pipetters and reagents.
  • Change gloves.
  • Aerosol barrier pipette tips.
  • Meticulous technique

10 bleach, acid baths, UV light
54
Uracil-N-Glycosylase (UNG)
  • Substitute dUTP for dTTP in initial PCR mix.
  • Proceed with PCR and detection.
  • In the next PCR to be set up, initial 30 min
    incubation at 37 C.
  • UNG will destroy any dUTP containing DNA products.

55
PCR Modifications
  • Nested PCR
  • Multiplex PCR
  • Tailed primers
  • Sequence-specific PCR
  • Allele specific
  • Reverse-transcriptase PCR
  • Long-range PCR
  • Whole-genome amplification
  • RAPD PCR (AP-PCR)
  • Quantitative real-time PCR

56
Reverse Transcription
  • Reverse transcription is the process through
    which a single-stranded RNA molecule gives rise
    to a complementary DNA (cDNA) molecule through a
    primer-dependent polymerase-dependent reaction.
  • First Strand Synthesis

57
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)
  • Steps in the RT-PCR reaction
  • RNA isolation
  • Reverse transcription
  • PCR amplification
  • Analysis of PCR product

58
Reverse Transcription of RNA (RT)
  • Primer Options for RT reaction
  • Oligo (dT)
  • Random Hexamers
  • Sequence-specific Primers
  • Enzyme Options for RT Reaction
  • Retroviral RNA-directed DNA polymerase
  • AMV Reverse Transcriptase (avian myeloblastosis
    virus)
  • MMLV Reverse Transcriptase (Moloney murine
    leukemia virus)

59
PCR Amplification Methods for Mutation Detection
  • Allele-specific amplification (uses
    sequence-specific primers for wild-type and
    mutant alleles)
  • Competitive oligonucleotide priming (uses
    sequence-specific primers for wild-type and
    mutant alleles that are present in the same
    reaction mixture)

60
Allele-specific PCR Amplification
61
PCR Applications
  • Structural analysis
  • DNA typing
  • Disease detection
  • Cloning
  • Mutation analysis
  • Detection of gene expression
  • Mapping
  • Site-directed mutagenesis
  • Sequencing

62
Analysis of PCR Products
63
REAL TIME PCR
  • Detects PCR products as they accumulate
  • Detect ds DNA by two methods
  • Intercalator fluorescent markers (ethidium
    bromide, syber green dye) non specific
  • Fluorogenic probes specific
  • Plot increase in fluorescence versus cycle number

64
GEL ANALYSIS VS FLUORESCENCE
65
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
66
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

67
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.

68
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.

69
Real Time PCR Growth Curve
70
Cycle Threshold (Ct)
71
Construction of Standard Curve
72
qPCR Detection Systems
  • DNA-specific dyes
  • Ethidium bromide
  • SyBr? green
  • Hybridization probes
  • Cleavage-based (TaqMan?)
  • Displaceable (Molecular Beacons?, FRET?)
  • Primer-incorporated probes

73
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

74
Cleavage-based Assay TaqMan 5-3 Exonuclease
Cleavage of Dual labeled Probe
75
Molecular Beacon Assay
76
FRET Probe
77
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

78
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.

79
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.

80
qPCR TaqMan?
81
qPCR FRET?
82
qPCR Molecular Beacons?
83
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
84
MOLECULAR AMPLIFICATION TECHNIQUES
  • Nucleic acid (NA) amplification methods fall into
    3 categories
  • Target amplification systems
  • Probe amplification systems
  • Signal amplification

85
Other Amplification Methods
  • Ligase chain reaction (LCR)
  • Branched DNA (bDNA)
  • Hybrid capture (HC)
  • Transcription-mediated amplification, self-
    sustaining sequence replication, nucleic acid
    sequence-based amplification (TMA, 3SR, NASBA)

86
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

87
Ligase Chain Reaction Amplification of Genomic DNA
  • Two primers are directed against adjacent target
    sequences.
  • Successfully annealed primers are ligated
    together through the action of a thermostable DNA
    ligase (amplification is accomplished through
    successive cycles of annealing and ligation).
  • Useful for detection of specific mutations in
    gene sequences.
  • Adapted for diagnostic testing of some infectious
    agents (chlamydia, gonorrhea, listeria, and HPV).

88
Ligase Chain Reaction
Template
Probes
...GTACTCTAGCT...
A G
T C
...CATGAGATCGA...
ligase

Target sequences are detected by coupled and
.
89
Ligase Chain Reaction Amplification of Genomic
DNA
90
Ligase Chain Reaction Mutation Detection
Utilizing Mutant-SpecificOligonucleotide Primers
91
LCR Detection ofChlamydia trachomatis
  • Cryptic plasmid target sequence (710 copies per
    organism)
  • 48 bp target within the cryptic plasmid
  • Unique DNA sequence (confers specificity)
  • Highly conserved among all C. trachomatis serovars

92
LCR Detection ofChlamydia trachomatis
  • Cryptic plasmid target sequence (710 copies per
    organism)
  • 48 bp target within the cryptic plasmid
  • Unique DNA sequence (confers specificity)
  • Highly conserved among all C. trachomatis serovars

93
Nucleic Acid Sequence Based Amplification (NASBA)
  • Reactions are isothermal (eliminating the need
    for a thermocycler).
  • All enzymatic reactions take place concurrently
    (reducing the total time to completion of
    procedure).
  • Provides exceptional sensitivity (109-fold
    amplification).
  • Applications include detection of HIV and other
    viruses (hepatitis, HTLV, CMV).
  • Available in kit form from
  • Organon Teknika (Durham, NC)

94
NASBAThe Basic Procedure
  • Hybridization of oligonucleotide-T7P primer to
    target sequence
  • Reverse transcription with reverse transcriptase
    (generation of RNADNA hybrid)
  • Digestion with RNase H

95
NASBAThe Basic Procedure (cont.)
  • Hybridization with target-specific
    oligonucleotide primer (P2)
  • Reverse transcription with reverse transcriptase
    (generation of double-stranded DNA)
  • Generation of RNA transcript by T7 RNA polymerase

96
NASBA
97
NASBA
98
NASBA
99
Transcription-Mediated Amplification (TMA)
  • RNA transcription amplification system utilizes
    two enzymes (RT and RNA pol).
  • Isothermal reaction, logarithmic amplification.
  • RNA or DNA targets.
  • Produces RNA amplicons.
  • Hybridization protection assay (HPA) simultaneous
    detection.

100
Microwell DNA Detection Systems
Addition of PCR Product (generated with
biotin-labeled primer)
Microwell with Bound Capture Probe
Colorimetric Detection
Addition of Avidin-enzyme Complexes
101
Strand Displacement Amplification
  • Strand displacement amplification and homogeneous
    real-time detection are incorporated in a
    second-generation DNA probe system, BDProbeTecET.
  • Little MC, et al.
  • Clin Chem 199945777784

102
Strand Displacement AmplificationThe
BDProbeTecET System
  • This system is based on the simultaneous
    amplification of nucleic acids by SDA and
    real-time detection using fluorescence energy
    transfer. It is useful in infectious disease
    testing (Chlamydia trachomatis and Neisseria
    gonorrhoeae).
  • High throughput
  • High sensitivity

103
Strand Displacement AmplificationInstrumentation
  • The BDProbeTecET instrument is a fluorescent
    reader capable of maintaining constant
    temperature, monitoring real-time fluorescence,
    and reporting results through an algorithm.
  • Additional instruments include heating blocks for
    sample preparation and priming steps of the
    reaction.

104
Strand Displacement AmplificationInstrumentation
  • Priming microwell contains dried SDA primers, one
    dNTP, and fluorescent oligonucleotide probe.
  • Amplification microwell contains the remaining
    dried SDA reagents, including enzymes.
  • SDA Reaction Phases
  • Target generation
  • Exponential target amplification
  • Detection

105
Strand Displacement AmplificationTarget
Generation
106
Strand Displacement AmplificationTarget
Generation
107
Strand Displacement AmplificationTarget
Generation
108
Strand Displacement AmplificationExponential
Target Amplification
109
Strand Displacement AmplificationExponential
Target Amplification
110
Strand Displacement AmplificationMechanism of
Fluorescence Energy Transfer
111
Strand Displacement AmplificationMechanism of
Fluorescence Energy Transfer
Fluorescein Label
Rhodamine Label

Dual-dye Labeled Hairpin Probe
Cleaved Duplex Yields Fluorescent Signal
BsoB1
112
Strand Displacement AmplificationDetection of
Neisseria gonorrhoeae
113
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.

114
Branched DNA Detection
115
Branched DNA (bDNA)
  • Isothermal
  • Signal amplification
  • A series of hybridizations attaches multiple
    signals to each target molecule.
  • HBV, HCV, HIV-1

116
Branched DNA
117
Hybrid Capture
  • Isothermal
  • Signal amplification
  • Immobilized DNA probes bind to RNA targets.
  • The RNADNA hybrids are bound by labeled
    monoclonal antibodies.
  • HPV, HBV, CMV

118
Hybrid Capture
119
Transcription-mediated Amplification (TMA)
  • Isothermal
  • Target amplification as RNA
  • cDNA is made from RNA target adding RNA
    polymerase promoter.
  • RNA is synthesized from the cDNA template and can
    serve as a source of new cDNA.
  • M. tuberculosis, C. trachomatis, HIV, CMV

120
Transcription-Mediated Amplification
121
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.

122
DNA Sequencing
123
Objectives
  • Compare and contrast the chemical (Maxam/Gilbert)
    and chain termination (Sanger) sequencing
    methods.
  • List the components and molecular reactions that
    occur in chain termination sequencing.
  • Discuss the advantages of dye primer and dye
    terminator sequencing.
  • Derive a text DNA sequence from raw sequencing
    data.
  • Describe examples of alternative sequencing
    methods, such as bisulfite sequencing and
    pyrosequencing.

124
Sequencing Methods
  • Maxam/Gilbert chemical sequencing
  • Sanger chain termination sequencing
  • Pyro-sequencing
  • Array sequencing

125
Maxam-Gilbert Sequencing

DMS
FA
H
HS
G
G
C
C
A
T
C
G
G
T
C
G
G
C
C
A
T
G
C
C
A
T
Maxam-Gilbert sequencing is performed by chain
breakage at specific nucleotides.
126
Maxam-Gilbert Sequencing

Sequencing gels are read from bottom to top (5'
to 3').
127
Chain Termination (Sanger) Sequencing
  • A modified DNA replication reaction.
  • Growing chains are terminated by dideoxy
    nucleotides.

128
Chain Termination (Sanger) Sequencing
  • The 3'-OH group necessary for formation of the
    phosphodiester bond is missing in ddNTPs.

Chain terminates at ddG
129
Chain Termination (Sanger) Sequencing
  • A sequencing reaction mix includes labeled primer
    and template.
  • Dideoxy nucleotides are added separately to each
    of the four tubes.

130
Chain Termination (Sanger) Sequencing

131
Chain Termination (Sanger) Sequencing
  • With addition of enzyme (DNA polymerase), the
    primer is extended until a ddNTP is encountered.
  • The chain will end with the incorporation of the
    ddNTP.
  • With the proper dNTPddNTP ratio, the chain will
    terminate throughout the length of the template.
  • All terminated chains will end in the ddNTP added
    to that reaction.

132
Chain Termination (Sanger) Sequencing
  • The collection of fragments is a sequencing
    ladder.
  • The resulting terminated chains are resolved by
    electrophoresis.
  • Fragments from each of the four tubes are placed
    in four separate gel lanes.

133
Chain Termination (Sanger) Sequencing

G A T C
Sequencing gels are read from bottom to top (5'
to 3').
134
Cycle Sequencing
  • Cycle sequencing is chain termination sequencing
    performed in a thermal cycler.
  • Cycle sequencing requires a heat-stable DNA
    polymerase.

135
Fluorescent Dyes
  • Fluorescent dyes are multi-cyclic molecules that
    absorb and emit fluorescent light at specific
    wavelengths.
  • Examples are fluorescein and rhodamine
    derivatives.
  • For sequencing applications, these molecules can
    be covalently attached to nucleotides.

136
Fluorescent Dyes
  • In dye primer sequencing, the primer contains
    fluorescent dyeconjugated nucleotides, labeling
    the sequencing ladder at the 5' ends of the
    chains.
  • In dye terminator sequencing, the fluorescent dye
    molecules are covalently attached to the dideoxy
    nucleotides, labeling the sequencing ladder at
    the 3' ends of the chains.

137
Dye Terminator Sequencing
  • A distinct dye or color is used for each of the
    four ddNTP.
  • Since the terminating nucleotides can be
    distinguished by color, all four reactions can be
    performed in a single tube.

A
The fragments are distinguished by size and
color.
T
G
T
138
Dye Terminator Sequencing
  • The DNA ladder is resolved in one gel lane or
    in a capillary.


G A T C


Slab gel
Capillary

139
Dye Terminator Sequencing
  • The DNA ladder is read on an electropherogram.

Capillary
Slab gel
Electropherogram

5' AGTCTG
140
Automated Sequencing
  • Dye primer or dye terminator sequencing on
    capillary instruments.
  • Sequence analysis software provides analyzed
    sequence in text and electropherogram form.
  • Peak patterns reflect mutations or sequence
    changes.

T/T T/A
A/A
5' AGTCTG
5' AG(T/A)CTG
5' AGACTG
141
Alternative Sequencing MethodsPyro-sequencing
  • Pyro-sequencing is based on the generation of
    light signal through release of pyrophosphate
    (PPi) on nucleotide addition.
  • DNAn dNTP ? DNAn1 PPI
  • PPi is used to generate ATP from adenosine
    phosphosulfate (APS).
  • APS PPI ? ATP
  • ATP and luciferase generate light by conversion
    of luciferin to oxyluciferin.

142
Alternative Sequencing MethodsPyro-sequencing

  • Each nucleotide is added in turn.
  • Only one of four will generate a light signal.
  • The remaining nucleotides are removed
    enzymatically.
  • The light signal is recorded on a pyrogram.

DNA sequence A T C A GG CC T
Nucleotide added A T C A G C T
143
Alternative Sequencing MethodsBisulfite
Sequencing
  • Bisulfite sequencing is used to detect
    methylation in DNA.
  • Bisulfite deaminates cytosine, making uracil.
  • Methylated cytosine is not changed by bisulfite
    treatment.
  • The bisulfite-treated template is then sequenced.

144
Alternative Sequencing MethodsBisulfite
Sequencing
  • The sequence of treated and untreated templates
    is compared.

GTC
GGC
GATCTATC
GTGCA

Me
Me
Me
Methylated
sequence
GTC
Treated sequence
Me
GGC
Me
GATUTATC
Me
GTGUA

DNA Sequence
(Untreated) reference ...GTCGGCGATCTATCGTGCA
Treated sequence
...GTCGGCGATUTATCGTGUA
This sequence indicates that these Cs are
methylated.
145
Summary
  • Genetic information is stored in the order or
    sequence of nucleotides in DNA.
  • Chain termination sequencing is the standard
    method for the determination of nucleotide
    sequence.
  • Dideoxy-chain termination sequencing has been
    facilitated by the development of cycle
    sequencing and the use of fluorescent dye
    detection.
  • Alternative methods are used for special
    applications, such as pyrosequencing (for
    resequencing and polymorphism detection) or
    bisulfite sequencing (to analyze methylated DNA).
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