Molecular Techniques II

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Molecular Techniques II
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Today

• Advanced PCR Techniques
• Other Amplification Technologies
• Primer/Probe Design
• Whole Genome/Transcriptome Amplification
• Post PCR Detection/Confirmation
• Molecular Typing Techniques
• Proteomic Techniques

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Advanced PCR Techniques

• qPCR methods
• Solid phase PCR
• ICC-PCR
• Long-Template PCR
• Control of Product Carryover

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qPCR Methods

• SyberGreen
• Minor Groove Binding Dyes
• Amplifluor Primers/LUX Primers
• FRET Technologies
• Taqman
• Molecular Beacons
• Hybridization Probes (HybProbes)
• Scorpion Primers

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Syber Green and Minor Groove Dyes

• Double Stranded DNA Binding Dyes
• Once Bound Fluorescence Increases
• Simplest technology, works with any primer set
• Non-specific
• Requires melting curve analyses or subsequent
  product analysis to confirm product

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Melt Curve Analysis
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Amplifluor and LUX Primers
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Taqman Probes
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Molecular Beacons
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Hybridization Probes
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Scorpion Primers
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Solid Phase PCR
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ICC-PCR

• Incorporates initial culture step into PCR
• More rapid than straight culture
• Better indication of infectivity than PCR alone
• Can alleviate some inhibition

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Long-Template PCR

• Another strategy for overcoming limitation of PCR
  to show viability
• Amplifies much longer section of target genome
• Difficult to optimize problems with secondary
  and tertiary structures
• Less efficient

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Control of Product Carryover

• Successful PCR can be your worst enemy
• Best control is structured work flow
• Other strategies
• UNG (uracil N-glycosylase)
• UV

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Other Nucleic Acid Amplification Strategies

• NASBA
• Rolling Circle

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NASBA
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Rolling Circle

• phi-29 DNA Polymerase
• Random Hexamers

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Primer and Probe Design

• For detection of organisms- Always a balance
  between specificity and sensitivity
• Dependent on target sequence and target structure
• Degenerate Primers
• Equimolar
• Universal base pairs
• Modifications
• Labels (fluorophores and biotin)
• Linkers
• Phosphorylation
• Modified bases (Universal, Ribobases, etc.)

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Whole Genome Amplification

• Strand Displacement
• GenomePlex Approach

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Multiple Strand Displacement
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GenomePlex Approach
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Post PCR Detection/Confirmation

• DNA Sequence Analysis
• Heteroduplex Mobility Assay
• Reverse Line Blot
• ELOSA

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DNA Sequence Analysis

• Gold Standard
• Essentially Reading of Amplified Genetic Code

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Heteroduplex Mobility Assay
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Reverse Line Blot
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Liquid Hybridization/ELOSA

• LH-Like Fluorescent Hybridization Assays, but
  typically Chemiluminescent
• ELOSA-Like ELIZA only using Oligonucleotides
  rather than Antibodies

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Molecular Typing Techniques

• RFLP/AFLP
• AP/RAPD PCR
• TRFLP

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RFLP
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AFLP
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RAPD-PCR
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TRFLP
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Proteomic Techniques

• MALDI-TOF MS
• SELDI-TOF MS

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MALDI-TOF MS
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SELDI-TOF MS
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Quantitation Considered

• Endpoint Dilution
• Quantal Assay/MPN
• Discrete Enumeration
• Fluorescent Detection

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End-Point Dilution

• Serial dilution (typically 10-fold)
• Presence/Absence or Discrete Enumeration
• Can be applied to most methods
• Robust, but subject to pipetting errors

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Quantal Assay/MPN

• Score each sample as /-
• Statistical estimation of titer
• Accuracy/Precision improves with increased
  replication
• Large confidence intervals

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Discrete Enumeration

• Direct count of Colonies/Plaques
• Accuracy/precision improves with replication
• Limited by concentration in counted dilution

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Fluorescent Detection

• Based on light emittance
• Luminometer
• Uses standard curves
• Indirect method (one more step to be inhibited)

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Detection Methods Compared

• Strengths?
• Weaknesses?
• Sensitivities?
• Specificity?