MLAB 2321 Molecular Diagnostics for Clinical Laboratory Science

Unit 9 Objectives- Nucleic Acid Amplification

  1. Name the individual who developed the polymerase chain reaction.
  2. List the 5 components required for the polymerase chain reaction and the function of each.
  3. Briefly describe the 3 steps in the polymerase chain reaction (PCR).
  4. State the optimal annealing temperature.
  5. State the optimal temperature of replication/primer extension.
  6. State the difference between the two-step PCR and three-step PCR.
  7. State the optimal length of primers.
  8. State the binding direction of the forward and reverse primers.
  9. Describe the problem of primer-dimer formation.
  10. List 6 potential sources of DNA template in a clinical sample.
  11. State the amount of DNA required for a successful PCR reaction.
  12. Describe the conditions which must be met for the best DNA template.
  13. State the source of Taq polymerase and describe the advantage of using it in a PCR procedure.
  14. State the source of Tth polymerase and describe the advantage of using it in a PCR procedure.
  15. List the components of the PCR buffer and the function of each.
  16. Describe a thermal cycler's function and the advantage this has over manual methods.
  17. State the importance of having a separate area for sample preparation, pre-PCR work, post-PCR work and analysis of results..
  18. State how the PCR product is analyzed.
  19. Given a PCR agarose gel identify: misprime, PCR product and primer dimer bands.
  20. List four controls which may be used to validate the PCR and the purpose of each.
  21. List and describe 4 methods used to control PCR contamination and state why this is so important.
  22. Define "mispriming" and breifly describe 3 hot-start set ups which may prevent or decrease this from occurring.
  23. BRIEFLY describe the following PCR modification procedures: Multiplex PCR, Reverse Transcriptase PCR, nested PCR, real time PCR and arbitrarily primed PCR.
  24. Briefly describe the principle of transcription-based amplification systems.
  25. Briefly describe the following probe amplification techniques: ligase chain reaction strand displacement amplification and QB replicase.
  26. Briefly describe the following signal amplification methods: branched DNA amplification, hbrid capture assays, cleavage based amplification and cycling probe.
  27. Define or describe the terms listed in the "Unit 9 Glossary of Terms ".

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