Today:• Advanced PCR Techniques• Other Amplification Technologies• Primer/Probe Design• Whole Genome/Transcriptome Amplification• Post PCR Detection/Confirmation• Molecular Typing Techniques• Proteomic Techniques
Advanced PCR Techniques
• qPCR methods
• Solid phase PCR
• ICC-PCR
• Long-Template PCR
• Control of Product Carryover
qPCR Methods
• SyberGreen• Minor Groove Binding Dyes• Amplifluor Primers/LUX Primers • FRET Technologies
– Taqman– Molecular Beacons– Hybridization Probes (HybProbes)– Scorpion Primers
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
ICC-PCR
• Incorporates initial culture step into PCR
• More rapid than straight culture
• Better indication of infectivity than PCR alone
• Can alleviate some inhibition
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
Control of Product Carryover
• Successful PCR can be your worst enemy
• Best control is structured work flow
• Other strategies– UNG (uracil N-
glycosylase)– UV
NASBA
3’3’5’5’
Primer 2
5’5’ 3’3’
5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’
3’3’5’5’3’3’
5’5’
RT 5’5’
3’3’5’5’
3’3’
3’3’5’5’
3’3’5’5’
5’5’3’3’
3’3’5’5’
5’5’3’3’
3’3’5’5’
Primer 1
Primer 2 RTRT
RT
RNase H
Cyclic PhaseCyclic Phase
T7 RNAPolymerase
5’5’3’3’
Primer 15’5’ 3’3’
5’5’ 3’3’3’3’
5’5’
RT
3’3’5’5’
RNase H
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.)
Post PCR Detection/Confirmation
– DNA Sequence Analysis– Heteroduplex Mobility Assay– Reverse Line Blot– ELOSA
Liquid Hybridization/ELOSA
• LH-Like Fluorescent Hybridization Assays, but typically Chemiluminescent
• ELOSA-Like ELIZA only using Oligonucleotides rather than Antibodies
Quantitation Considered
• Endpoint Dilution
• Quantal Assay/MPN
• Discrete Enumeration
• Fluorescent Detection
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
Quantal Assay/MPN
• Score each sample as +/-• Statistical estimation of titer• Accuracy/Precision improves with increased
replication• Large confidence intervals
Discrete Enumeration
• Direct count of Colonies/Plaques
• Accuracy/precision improves with replication
• Limited by concentration in counted dilution
Fluorescent Detection
• Based on light emittance
• Luminometer
• Uses standard curves
• Indirect method (one more step to be inhibited)