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Results

Our project focused on developing an isothermal, point-of-care diagnostic method for detecting EGFR exon 19 deletion mutations in circulating tumor DNA (ctDNA) using Recombinase Polymerase Amplification (RPA). Over four design cycles, we optimized our amplification, purification, and verification processes.

Amplification of Target ctDNA

Through multiple rounds of design, testing, and refinement, we successfully demonstrated that RPA can amplify the EGFR mutation sequence, confirming the feasibility of isothermal amplification for ctDNA detection.

Among all primers tested, the 5 bp overlap forward primer (BBa_25ZSP4AQ) produced the most specific and intense band, confirming its strong performance in mutation detection.

RPA Amplification Gel 1 RPA Amplification Gel 2

Gel electrophoresis from Test 1 showing RPA amplification with all primers, with the 5 bp overlap forward primer showing the highest band intensity.

However, some off-target amplification was observed, producing unexpected bands around 500 bp. Subsequent tests confirmed that this was not the input DNA but potential off-target amplification or contamination inherent to RPA.

Improving Results Clarity

To improve gel results, we implemented a purification step using the PureLink PCR Purification Kit, which successfully removed protein–DNA complexes and reduced background smears, resulting in clearer and sharper gel bands.

Sample order in the gel:

  1. 100–300 bp DNA ladder
  2. RPA sample with 0 ng of mutated DNA template input
  3. RPA sample with 0.5 ng of mutated DNA template input
  4. RPA sample with 1 ng of mutated DNA template input
  5. RPA sample with 5 ng of mutated DNA template input
  6. DNA input (5 bp overlap forward primer, reverse primer, mutated DNA template input)
  7. 100 ng of wildtype DNA template
  8. 100 ng of mutated DNA template

Purified Gel Gel electrophoresis after PCR purification showing sharper bands.

While contamination remained an issue in RPA, switching to PCR verification showed that our mutation primers consistently amplified the correct target region with clean single bands and no contamination. Sanger sequencing further confirmed that the amplified fragment matched the target mutation region. However, the primers did not reach the desired specificity to the mutation and still amplified the wildtype input sequence.

Gel lane order (left to right): Each group consists of the same primer pair tested with wildtype template, mutation template, and a negative control (water). Groups differ by annealing temperature as follows:

  1. Wildtype forward primer + reverse primer (72°C)
  2. 4 bp overlap primer + reverse primer (72°C)
  3. 5 bp overlap primer + reverse primer (71°C)
  4. 6 bp overlap primer + reverse primer (70°C)
  5. 8 bp overlap primer + reverse primer (72°C)
  6. Forward template primer + reverse template primer (61°C)

PCR Verification Gel PCR gel showing mutation and wildtype amplification.

Summary of Findings

  • RPA successfully amplified the mutation sequence in EGFR exon 19 under isothermal conditions.
  • The 5 bp overlap primer showed the best performance in terms of specificity and amplification yield.
  • Purification significantly improved gel clarity and reliability.
  • PCR and sequencing verification confirmed the accuracy of the amplified mutation region.

Next Steps

To further enhance specificity and reduce false positives, future work will focus on:

  • Integrating CRISPR-Cas9 processing prior to amplification to remove wildtype interference.
  • Developing a Lateral Flow Assay (LFA) for visual readout, enabling a complete one-pot, field-deployable diagnostic kit.