Molecular Techniques II

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

Melt Curve Analysis

Amplifluor and LUX Primers

Taqman Probes

Molecular Beacons

Hybridization Probes

Scorpion Primers

Solid Phase PCR

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

Other Nucleic Acid Amplification Strategies NASBA Rolling Circle

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

Rolling Circle phi-29 DNA Polymerase Random Hexamers

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.)

Whole Genome Amplification Strand Displacement GenomePlex Approach

Multiple Strand Displacement

GenomePlex Approach

Post PCR Detection/Confirmation –DNA Sequence Analysis –Heteroduplex Mobility Assay –Reverse Line Blot –ELOSA

DNA Sequence Analysis Gold Standard Essentially Reading of Amplified Genetic Code

Heteroduplex Mobility Assay

Reverse Line Blot

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

Molecular Typing Techniques –RFLP/AFLP –AP/RAPD PCR –TRFLP

RFLP

AFLP

RAPD-PCR

TRFLP

Proteomic Techniques MALDI-TOF MS SELDI-TOF MS

MALDI-TOF MS

SELDI-TOF MS

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)

Detection Methods Compared Strengths? Weaknesses? Sensitivities? Specificity?