Week 6 HW

What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

A Phusion HF PCR Master Mix is a pre-combined PCR reaction system optimised for a specific engineered DNA polymerase.

  • Phusion DNA polymerase — provides the catalytic activity which synthesises DNA, and includes a 3’→5’ exonuclease proofreading to reduce error
  • Reaction buffer
  • MgCl₂ — magnesium ions
  • dNTPs — deoxynucelotide triphosphates: dATP, dCTP, dGTP, and dTTP
  • Stabilizers/additives
  • Water

A typical setup only requires after adding:

  • Forward primer
  • Reverse primer
  • Template DNA
  • Additional water to reach final volume

What are some factors that determine primer annealing temperature during PCR?

Factors:

  • Primer melting temperature — dominant factor
  • GC content of primer
  • Primer length
  • Sequence features
  • Salt concentration in the reaction buffer
  • Template–primer mismatch tolerance

There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other.

PCR creates DNA fragments by enzymatic replication using primers that define the fragment boundaries.

Protocol: The reaction contains template DNA, forward and reverse primers, dNTPs, buffer, Mg²⁺, and a thermostable DNA polymerase (e.g. Phusion or Taq). The protocol cycles temperature: denaturation (~95 °C) separates strands, annealing (~50–65 °C) allows primers to bind, and extension (~72 °C) synthesizes new DNA. After ~25–35 cycles, the region between the primers is exponentially amplified, producing many linear copies of a precisely defined sequence.

Restriction enzyme digestion produces linear fragments by cutting DNA at specific recognition sequences using restriction endonucleases.

Protocol: The protocol involves incubating DNA with one or more enzymes in the appropriate buffer (often ~37 °C) for a set time. The enzyme recognizes a short sequence (typically 4–8 bp) and cleaves the phosphodiester backbone, generating fragments with defined ends (blunt or sticky). The resulting fragment sizes depend entirely on where those recognition sites exist in the DNA.

Conceptually, PCR synthesizes a fragment by copying between two designed boundaries, whereas restriction digestion extracts a fragment by cutting an existing molecule at predetermined sequence motifs.

To understand when both are useful, consider an objective: engineer E. coli to produce human insulin, which requires building a plasmid containing the insulin gene under a bacterial promoter.

3 difference scenarios for getting insulin:

  1. DNA comes from a biological sample (e.g. human genomic DNA). The insulin gene is buried inside billions of unrelated bases, so PCR is used to isolate and amplify only that specific region using primers that define its boundaries. PCR is therefore used when the goal is to retrieve a specific gene from a complex DNA mixture.

  2. DNA already exists in a plasmid (e.g. moving GFP from plasmid A into plasmid B). The fragment is already isolated, so restriction enzymes are used to cut DNA at specific recognition sequences, allowing the gene to be excised and inserted into another vector. Restriction digestion is therefore used when the task is to cut and rearrange existing DNA molecules.

  3. DNA is chemically synthesized because the sequence is already known. The synthesized fragment may still be PCR-amplified if more copies are needed, and restriction enzymes (or similar assembly methods) are used to insert it into plasmids. In practice, PCR isolates or amplifies sequences, while restriction enzymes cut DNA molecules so fragments can be inserted, removed, or reorganized.

How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

Desiderata: Linear DNA fragments whose terminal 20–40 bp regions are perfectly homologous to the neighboring fragment, unique, structurally stable, and present in a clean preparation so the Gibson enzymes can expose the overlaps, allow annealing, fill gaps, and ligate the final construct.

How does the plasmid DNA enter the E. coli cells during transformation?

A cell is boundaried by a lipid membrane wall. It is not a solid wall. It is more of a dense molecular fluid. Each phospholipid is held in place only by weak interactions (hydrophobic forces, van der Waals forces). Like an electron has no fixed static wall, rather a field it creates, a cell has no fixed solid wall, it is a highly dense molecular fluid.

Lipids constantly fluctuate, small gaps appear and disappear.

During transformation, the culture is treated to brief heat shock (~42 °C for ~30–60 s). The rapid temperature change causes a sudden increase in the lipid kinetic energy, resulting in transient disordering of phospholipid packing, resulting in transient aqueous pores in bilayer. Plasmid DNA molecules enter through these pores.

This is paired with a treatment of calcium ions, which neutralises negative charges on the DNA phosphate backbone and the membrane surface, and thus reduces electrostatic repulsion between DNA and cell envelope.

Describe another assembly method in detail (such as Golden Gate Assembly)

Explain the other method in 5–7 sentences plus diagrams (either handmade or online).

Design fragments with Type IIS sites and specific 4-bp overhangs. PCR amplify or synthesize fragments with those flanking sites. Mix fragments, plasmid backbone, Type IIS enzyme, ligase, and buffer. Run digestion–ligation thermal cycles. Transform assembled plasmid into bacteria.

Model this assembly method with Benchling or Asimov Kernel!