Week 6 HW: DNA Assembly

DNA Assembly

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

Phusion High-Fidelity PCR Master Mix contains Phusion DNA Polymerase, dNTPs, reaction buffer, and MgCl₂. The DNA polymerase copies the DNA template during PCR. dNTPs are the building blocks used to synthesize the new DNA strand. The buffer keeps the reaction at the correct pH and salt condition. MgCl₂ is an important cofactor that helps the polymerase function properly. Since Phusion is a high-fidelity polymerase, it is useful when the amplified DNA sequence needs to be accurate for cloning or assembly.

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

Primer annealing temperature mainly depends on the primer’s melting temperature, or Tm. Tm is affected by primer length, GC content, sequence composition, and whether the primer has mismatches with the template. Longer primers and higher GC content usually increase the Tm. Salt concentration and buffer conditions can also affect annealing. In practice, the annealing temperature should be high enough to avoid nonspecific binding, but not so high that the primer cannot bind efficiently.

3. Compare PCR and restriction enzyme digests as ways to create linear DNA fragments.

PCR creates linear DNA fragments by amplifying a selected region from a DNA template using primers. It is flexible because primers can be designed to add extra sequences, such as Gibson overlaps, mutations, or restriction sites. Restriction enzyme digest creates linear fragments by cutting DNA at specific recognition sites. It is simpler when useful cut sites already exist in the plasmid or insert, but it is limited by where those enzyme sites are located. PCR is preferable when custom fragment boundaries or added overlap sequences are needed. Restriction digest is preferable when the construct already has clean enzyme sites and a more direct cut-and-paste workflow is possible.

4. How can you ensure that digested or PCR-ed DNA is appropriate for Gibson cloning?

For Gibson cloning, each neighboring DNA fragment needs compatible overlapping ends. Primers can be designed so that PCR products contain overlap regions matching the adjacent fragment or vector. The overlap should be long enough for efficient assembly, usually around 15–30 bp depending on the protocol and sequence composition. The fragments also need to be in the correct order and orientation, without unwanted mutations. The vector should be linearized only once. After PCR or digestion, fragment size can be checked by gel electrophoresis, and the final assembled construct should ideally be confirmed by sequencing.

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

During transformation, plasmid DNA enters chemically competent or electrocompetent E. coli cells. In heat-shock transformation, calcium chloride treatment helps make the cell membrane more permissive to DNA, and the short heat shock creates a temporary condition that allows plasmid DNA to pass into the cells. In electroporation, an electrical pulse briefly opens pores in the bacterial membrane, allowing DNA to enter. After transformation, the cells recover in growth medium and are plated on antibiotic plates. Only the cells that successfully took up the plasmid with the antibiotic resistance marker should grow.

6. Golden Gate Assembly.

Golden Gate Assembly is a DNA assembly method that uses Type IIS restriction enzymes and DNA ligase in the same reaction. Type IIS enzymes, such as BsaI or BsmBI, recognize a specific DNA sequence but cut outside of that recognition site. This allows the designer to create custom sticky ends, or overhangs, that determine the order of DNA fragments. After the enzyme cuts the fragments, T4 DNA ligase joins compatible overhangs together. Because the recognition sites can be designed to disappear from the final product, Golden Gate can produce scarless assemblies. It is especially useful for modular assembly of multiple DNA parts, such as promoter, RBS, coding sequence, and terminator. Unlike Gibson Assembly, which relies on longer homologous overlaps, Golden Gate relies on short designed overhangs produced by restriction enzyme cutting.