1. Molecular Techniques II

2. Today: Advanced PCR Techniques Other Amplification Technologies Primer/Probe Design Whole Genome/Transcriptome Amplification Post PCR Detection/Confirmation Molecular Typing Techniques Proteomic Techniques

3. Advanced PCR Techniques qPCR methods Solid phase PCR ICC-PCR Long-Template PCR Control of Product Carryover

4. qPCR Methods SyberGreen Minor Groove Binding Dyes Amplifluor Primers/LUX Primers FRET Technologies –Taqman –Molecular Beacons –Hybridization Probes (HybProbes) –Scorpion Primers

5. 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

6. Melt Curve Analysis

7. Amplifluor and LUX Primers

8. Taqman Probes

9. Molecular Beacons

10. Hybridization Probes

11. Scorpion Primers

12. Solid Phase PCR

13. ICC-PCR Incorporates initial culture step into PCR More rapid than straight culture Better indication of infectivity than PCR alone Can alleviate some inhibition

14. 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

15. Control of Product Carryover Successful PCR can be your worst enemy Best control is structured work flow Other strategies –UNG (uracil N- glycosylase) –UV

16. Other Nucleic Acid Amplification Strategies NASBA Rolling Circle

17. 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

18. Rolling Circle phi-29 DNA Polymerase Random Hexamers

19. 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.)

20. Whole Genome Amplification Strand Displacement GenomePlex Approach

21. Multiple Strand Displacement

22. GenomePlex Approach

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

24. DNA Sequence Analysis Gold Standard Essentially Reading of Amplified Genetic Code

25. Heteroduplex Mobility Assay

26. Reverse Line Blot

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

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

29. RFLP

30. AFLP

31. RAPD-PCR

32. TRFLP

33. Proteomic Techniques MALDI-TOF MS SELDI-TOF MS

34. MALDI-TOF MS

35. SELDI-TOF MS

36. Quantitation Considered Endpoint Dilution Quantal Assay/MPN Discrete Enumeration Fluorescent Detection

37. 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

38. Quantal Assay/MPN Score each sample as +/- Statistical estimation of titer Accuracy/Precision improves with increased replication Large confidence intervals

39. Discrete Enumeration Direct count of Colonies/Plaques Accuracy/precision improves with replication Limited by concentration in counted dilution

40. Fluorescent Detection Based on light emittance Luminometer Uses standard curves Indirect method (one more step to be inhibited)

41. Detection Methods Compared Strengths? Weaknesses? Sensitivities? Specificity?