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Published byCharlene Dickerson Modified over 9 years ago
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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 3’ 5’ Primer 2 5’3’ 5’3’5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ RT 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’5’ 5’ 3’ 3’ 5’ Primer 1 Primer 2 RT RT RNase H Cyclic Phase T7 RNA Polymerase 5’ 3’ Primer 15’ 3’ 5’ 3’ 3’ 5’ RT 3’ 5’ RNase H
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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?
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