Mutagenesis & protein engineering 1) Understand how changes can be incorporated
ID: 193255 • Letter: M
Question
Mutagenesis & protein engineering 1) Understand how changes can be incorporated into PCR primers 2) Random methods: a) b) c) How does error-prone PCR introduce variation? What is meant by a degenerate primer? What method can you use if you have a panel of similar sequences you want to recombine? 3) Directed evolution a) Why are the following areas important: i) Generating sequence diversity? Connecting proteins to their encoding DNA sequence? ii) Selections and screens for activity? Why is colony 'picking' a low-throughput method? Why are colorimetric or fluorescence assays desirable? i) How was the GloSensor in Lecture 9 designed? b) c) d) Display technologies Yeast surface display (1) Which of the 3 points in 4.a does the yeast cell itself solve? (2) How can fluorescently tagged proteins be used with FACS to sort yeast cells? i) ii Other displays (1) Why can't phage display be used for FACS screens? (2) How can phage display libraries be screened for protein-protein interactions?Explanation / Answer
1) PCR primers can be subjected to site directed mutagenesis. This involves incorporating mutagenic internal primers. Hybridized primers are produced in the first round and primers with mutated internal sequence are produced in the second round of PCR.
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