Challenge 3: How Can We Amplify Our Signal?

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.

Design

• Using this sequence from the SARS-CoV-2 Nucleocapsid gene, design a set of primers to perform an RT-PCR assay:
  • 5’-GACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGA-3’
  • 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.
  

Calculate

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

Graph

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

Write

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

Share and Discuss

• Your results.