https://pages.htgaa.org/2026a/grace-hussey/homework/week-06-hw-genetic-circuits-part-i/index.htmlDNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? DNA polymerase: uses dNTP monomers to synthesize new DNA strands dNTPs: monomers of new DNA strand Buffer: stabilizes the pH of the reaction for optimal enzymatic function MgCl2: Cofactor for the polymerase; optimizes enzymatic function and primer annealing What are some factors that determine primer annealing temperature during PCR? GC content (higher GC content = more hydrogen bonds = stronger primer annealing = higher annealing temperature) Primer length (longer primers = more hydrogen bonds = stronger primer annealing = higher annealing temperature) 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: Enzyme: DNA polymerase DNA polymerases bind to primers and synthesize complementary DNA to the template strand Purpose: amplify! Polymerases synthesize DNA by recognizing primers (designed to flank the specific region of interest) and incorporating dNTPs into a novel DNA strand When to use: To detect a specific sequence within a mixed sample To create more of a specific DNA sequence (amplify) Restriction enzyme: Enzyme: restriction endonuclease Restriction endonucleases recognize specific nucleotide sequences (4-8 bp) in double-stranded DNA and cut in a specific pattern (blunt or sticky ends, depending on the enzyme) Purpose: cut! Restriction enzymes cut existing template DNA, they do NOT amplify DNA fragments When to use: To linearize bacterial plasmids (ex. for in vitro transcription of capped mRNA for microinjection into X. laevis!) To cut DNA fragments to assemble/ligate together and transform into a plasmid How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? Primer design! The 5’ tail overhang of each primer should be identical to the adjacent DNA fragment the 3’ end of the primer should be complementary to the DNA fragment to which it will anneal Restriction Digest: Restriction enzyme cut sites should be avoided PCR: Primers should be designed that flank the overhang regions How does the plasmid DNA enter the E. coli cells during transformation? Heat shock transformation - briefly raising temperature increases the permeability of the bacterial cell wall, allowing the plasmid DNA to enter into the E. coli cell E. coli cell sample placed on ice for ~15-30 minutes Sample heated to 42ºC for ~15-30 seconds Sample then returned to ice for ~5 minutes Describe Golden Gate Assembly Golden Gate Assembly utilizes Type IIS restriction enzymes (such as BsaI) which recognize non-pallindromic sequences, cut outside the recognition site (to avoid damaging the DNA sequence of interest), and create sticky ends of variable lengths. First, template DNA is amplified using PCR with primers specified to include the TIIS recognition and cut sites. Next, a restriction digest with the TIIS enzyme is performed to create DNA fragments with sticky ends. The sticky ends of adjacent fragments should be complementary so the sequences can be ligated in the appropriate order. Following ligation of DNA fragments with a plasmid, the engineered plasmid can be transformed into a bacterial cell (ex. E. coli), and bacterial colonies containing the plasmid can be screened for furhter use. Image generated by ChatGPTHugoen