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

3’3’5’5’

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

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

TR

FLP

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?