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Challenge 3: How Can We Amplify Our Signal?

Learning Targets:

  • I can explain and use amplification.
  • I can model amplification.

Activity 1: How does amplification work?

Answer the following:

Our sample may contain very few viruses and our test may not be sensitive enough to detect the presence of the virus RNA or protein. How can we design a test that increases the signal? How can we increase the amount of viral nucleic acid?

  • Is there a way to copy nucleic acids?
    • What do our cells need to do before dividing?
  • Describe the process for how our cells copy their DNA.
  • Could we use a similar process to copy viral RNA?
    • How can we convert viral RNA into DNA?
      • Reverse transcriptase (RT)
      • Oligomer that anneals to our RNA
    • Can we repeat the process to make several copies of the DNA?
      • Polymerase chain reaction (PCR)
    • How many new DNA copies are produced after each round of PCR?

Share & Discuss

  • Your responses.

Activity 2 - Design a RT-PCR Assay


  • Using this sequence from the SARS-CoV-2 Nucleocapsid gene, design a set of primers to perform an RT-PCR assay:
    • Primers should be 20 nucleotides in length
      • Forward primer: 5’-GAC CCC AAA ATC AGC GAA AT-3’
      • Reverse primer: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’

Outline a protocol for performing RT-PCR

  • Reverse Transcription Step
    • Purified RNA from our sample
    • Reverse primer
    • Reverse transcriptase
    • Nucleotides
  • Polymerase Step
    • Sample from Reverse Transcription Step
    • Reverse primer
    • Forward primer
    • TAQ polymerase (A thermostable DNA polymerase)
    • Nucleotides

Share & Discuss

  • Examine each other’s designs, offering helpful suggestions if needed.

Activity 3: Using PCR


  • The number of DNA molecules after 30 rounds of PCR. Assume our sample has 10 molecules of SARS-CoV-2 nucleocapsid RNA.


  • The number of DNA molecules after each of the first 5 rounds of PCR. How would you characterize the line?


  • A formula that would allow you to estimate the number of DNA molecules for any number of PCR cycles.

Share and Discuss

  • Your results.