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Molecular Techniques II

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

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Molecular Techniques II

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  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. 5’ 3’ Cyclic Phase Primer 1 5’ 3’ 5’ 3’ 3’ 5’ 3’ RT RT 5’ Primer 2 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ RNase H 5’ 3’ RNase H 5’ Primer 2 3’ 5’ Primer 1 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ RT RT 5’ 5’ 3’ 3’ 3’ T7 RNA Polymerase 3’ 5’ 5’ NASBA

  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?

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