Lecture 4

Question 1

What are the steps of Error Prone PCR/DNA shuffling

Answer 1

1) Generate library of DNA constructs
2) Express all constructs
3) Screen protein with designed characteristcs
4) Large scale expansion

Question 2

What are the steps of Directed evolution

Answer 2

1) Original gene
2) Mutant gene
3) Transfection
4) Protein purification
5) Screening
6) Sequencing of proteins showing good activity

Question 3

How can you achieve error prone PCR when you have low fidelity

Answer 3

• Increase Mg2+ conc
• Addition of Mn2+
• Unequal concs of 4 dNTPs
• Use dINP
• Use more Taq polymerase

Question 4

What is gene shuffling

Answer 4

Generation of random fragments by DNase I followed by the reassembly of those fragments into a full length gene by primerless PCR. Chimeric genes expressed in a plasmid

Question 5

What is RFLP analysis (in regards to gene shuffling)

Answer 5

Use restriction enzymes to create smaller fragments. There is a reduced number of parental forms being reformed.