Course 4

Polymerase Chain Reaction (PCR) Technology (14 hours)

This course is suitable for school-level, undergraduate and graduate students, and those engaged in scientific research, diagnostics and teaching which involves Polymerase Chain Reaction (PCR) Technology. 

Theory:

 

  • Guidelines for PCR

  • Principle of PCR

  • Critical factors for successful PCR

  • Designing PCR primers

  • Standard practices in a PCR Laboratory

  • Troubleshooting and prevention of carryover contamination

  • Optimization strategies for PCR

 

PCR product analysis:

 

  • Detection of PCR products by gel electrophoresis

  • Methods of purification and quantification of PCR products

  • Methods of labelling of PCR products (PCR probes) and visualization of label

  • Cloning of PCR products: TOPO cloning

  • DNA sequence analysis of PCR product 

 

PCR Applications:

 

  • Restriction Fragment Length Polymorphism (RFLP) and Randomly Amplified Polymorphic DNA (RAPD) analysis

  • Short Tandem Repeat (STR) (micro-satellite) analysis

  • Multiplex PCR

  • Reverse Transcription (RT)-PCR

  • PCR-ELISA (Enzyme Linked Immunosorbent Assay)

  • In-situ PCR

  • Quality Control/Quality Assurance of PCR assays

Practicals:

 

  • Extraction of DNA from cells, blood stains for PCR

  • A standard PCR assay

  • Analysis of PCR products by agarose and acrylamide gel electrophoresis

  • Strategies for optimization of PCR

  • Purification of PCR products

  • Labelling of PCR products

  • Primer design using online sequence databases

  • PCR-RFLP assay

  • Multiplex PCR assay

  • RT-PCR assay

  • PCR-ELISA assay

  • PCR troubleshooting

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