Real-Time PCR

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Coauthors: Heather MacDonald, M(ASCP), MB(ASCP) and Ryanne Wolfe, MS, MBA
Reviewer: Marissa Wildung, MLS(ASCP)CM

This course provides a brief overview of DNA and RNA structure and the principles of polymerase chain reaction. It then proceeds to discuss the principles and procedures for real-time PCR, highlighting the advantages over traditional PCR methods and the applications in the clinical setting.

Continuing Education Credits

Objectives

  • Compare and contrast the structure and functional regulation of DNA and RNA.
  • Discuss the basic principles of the polymerase chain reaction (PCR) process.
  • Define the detection techniques for the measurement of nucleic acids by real-time PCR.
  • Discuss the evolution of traditional PCR to real-time PCR, and the advantages and limitations of both methods.
  • State the applications of real-time PCR.

Course Outline

  • Historical Landmarks in Molecular Diagnostics
      • Historical Landmarks in Molecular Diagnostics
  • Composition and Formation of DNA and RNA
      • Molecular Composition of DNA
      • Molecular Composition of RNA
      • Formation of DNA and RNA Strands
      • Which of the following characteristics is common to both RNA and DNA?
      • True or False: The DNA strand ending with a phosphate group is the 5' end.
  • Extraction and Isolation of Genetic Material
      • Nucleic Acid Isolation
      • Automated vs. Manual Extraction
      • There are several methods for extracting nucleic acids (DNA and/or RNA) from samples. The most common protocols are manual or automated which essentia...
  • Introduction to Polymerase Chain Reaction (PCR)
      • Introduction to PCR
      • Steps of PCR: Denaturation
      • Steps of PCR: Annealing (Hybridization)
      • Steps of PCR: Extension
      • Repetitive Cycling
      • Overview of PCR Basic Principles
      • What is the first step of the PCR reaction?
      • Match the names of the PCR stages to their definitions.
  • Real-Time PCR
      • Conventional PCR vs. Real-Time PCR
      • Match each characteristic listed below to either conventional or real-time PCR.
      • Real-Time PCR Components
    • Primers
      • Primers
    • Polymerase
      • Polymerase
    • Enzyme Cofactors, dNTPs, and Salts/Buffers
      • Enzyme Cofactors, dNTPs, and Salts/Buffers
      • Reverse Transcriptase PCR (RT-PCR)
    • Thermocyclers and Detection Methods
      • Thermocyclers
      • Detection Methods
      • Phases of Real-Time PCR
      • During which phase in real-time PCR can the fluorescent signal start to be recorded?
      • True or False: Thermocyclers are instruments used to amplify DNA and RNA samples by the polymerase chain reaction.
  • Applications of Real-Time PCR
      • Laboratory Applications
      • Multiplexing
      • Microbe Detection
      • SNP Detection
      • Viral Load
      • True or False: One of the best ways to evaluate single nucleotide polymorphism (SNP) is by melting curve analysis.
      • What is the PCR type used to detect two or more target sequences of DNA or RNA simultaneously in a single sample called?
  • Conclusion
      • Conclusion
  • References
      • References

Additional Information

Level of Instruction: Intermediate
Intended Audience: Medical laboratory scientists, medical technologists, and technicians, This course is also appropriate for clinical laboratory science students and pathology residents.
Author Information:
Heather MacDonald, M(ASCP), MB(ASCP) has over 10 years of clinical laboratory experience and oversees a Molecular Diagnostics Laboratory. She is currently the Advanced Diagnostics Manager for Children's Healthcare of Atlanta in Georgia. Along with performing routine diagnostic assays, implementing laboratory-developed qualitative and quantitative molecular assays (both singleplex and multiplex) is her primary focus. Heather has published numerous articles and presents her research at national and international meetings. She has also worked with numerous corporations to bring commercial assays to market.
Ryanne Wolfe holds an MS in biotechnology and an MBA from the University of Maryland. Ryanne has worked in the healthcare field for eight years, most recently in the laboratory at St. Christopher’s Hospital for Children in Philadelphia, Pennsylvania.
Reviewer Information: Marissa Wildung, MLS(ASCP)CM, is a Laboratory Supervisor with nine years of experience as a Medical Laboratory Scientist and a Faculty Instructor for an MLT/Phlebotomy Program in Central Minnesota. Marissa holds a Bachelor’s in Medical Laboratory Science from the University of Cincinnati and is pursuing a Master's in Business Administration from St. Cloud State University.
Course Description: This course provides a brief overview of DNA and RNA structure and the principles of polymerase chain reaction. It then proceeds to discuss the principles and procedures for real-time PCR, highlighting the advantages over traditional PCR methods and the applications in the clinical setting.

This course is part of:
Compliance & CE
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