BioSecurity Twist Problem 1

Members: Tom Stoughton, Susi Franco, Sameer Bhalesha, Bhavitha Kandru, Sumanth W

Our Problem

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Problem Statement: The problem at hand is to design and develop an open-source software tool capable of analyzing an oligo pool of any size and identifying potential combinations of oligos in that pool that could be assembled using restriction, PCA, Gibson-like methods or other assembly protocol. This tool aims to enhance biosecurity by ensuring that oligo pools cannot be exploited to assemble sequences that might pose risks or otherwise be subject to regulatory control.

What is Oligo?

• Oligos are short, synthetic strands of DNA or RNA.
• 3 methods to create them: Restriction, PCR, Gibson-like

What is the three methods to combine oligos?

• Restriction: A method which uses restriction enzymes to cut DNA at specific sites so that they can be joined together in a new order.
  • Regarding Oligos:
    • Oligos are designed to have restriction enzyme recognition sites at their ends. This allows them to be "cut" by the enzymes and then "stitched" together with other DNA fragments that have complementary overhangs.
    • Oligos can also be used to introduce specific sequences or mutations at the restriction sites, making this method useful for targeted gene editing or cloning.
    • Because each restriction enzyme recognizes and cuts at a specific sequence, the design of oligos must be precise to ensure the desired assembly.
• PCA (Polymerase Cycling Assembly): A method that uses cycles of denaturing, annealing, and extending to assemble overlapping DNA fragments into a full-length sequence.
  • Regarding Oligos:
    • Oligos used in PCA need to have overlapping sequences with the adjacent DNA fragments. These overlaps guide the correct assembly order.
    • During the cycling process, DNA polymerases extend the overlapping regions, stitching the fragments together.
    • The oligos serve as primers for this extension process, and as the cycles proceed, the full-length assembled sequence becomes the dominant product.
    • The length and quality of the overlap, which is part of the oligo design, play a crucial role in the efficiency of PCA.
• Gibson-like: Refers to the Gibson Assembly method, which is a way to join multiple fragments of DNA together in a specific order without the use of restriction enzymes. Instead, it relies on overlapping sequences and a cocktail of enzymes to stitch them together seamlessly.
  • Regarding Oligos:
    • Like PCA, Gibson Assembly relies on overlapping sequences between DNA fragments for correct assembly. Oligos are designed to have these overlaps.
    • During the assembly, exonucleases in the enzyme mix chew back the ends of the DNA fragments, exposing the overlapping sequences. Other enzymes then fill in gaps and ligate the fragments together.
    • This method allows for seamless assembly, meaning there are no extra nucleotides or scars at the junctions. The quality and length of the overlap (part of the oligo design) are vital for efficient assembly.

Which method could create the most dangerous molecules with oligos?

Restriction Assembly: