Site Directed Mutagenesis

Question 1

what are the draw backs of random mutagenesis?

Answer 1

▪ Non-specific
▪ Low frequency of mutants for one gene (among thousands)
▪ Possible multiple mutations in different genes
▪ Uncertainty about phenotype-genotype relationship

Question 2

what is the classical method of SDM?

Answer 2

requires a single primer, ssDNA template, klenow DNA pol (no 5’3’ exonuc activity);p-32 oligonuc to detect mutants

• -select clones
• -verify

Question 3

disadvantages of M13 based SDM?

Answer 3

need ssDNA
low mutant:WT –>theoretical max of 50:50, but much lower in practice
DNA pol (klenow or T7) is low fidelity, not thermostable
-use radioactive P-32

Question 4

what are the commercial kits available for SDM?

Answer 4

quikchange
phusion
genetart

Question 5

advantage of thermo scientific fusion kit?

Answer 5

amplification is exponential

primers do not overlap

Question 6

problems with TS phusion kit?

Answer 6

after amplification, have blunt end; ligation of blunt end not efficient; recircularizing plasmid means you cannot have too much DNA; plasmids will link to other plasmids

Question 7

what is the overlap extension methods?

Answer 7

have2 mutagenic primers and 2 non mut; one F and one R; synthesizes; denature and overlap mutated DNA; –>synthesize; just like overlap PCR

Question 8

check gibson assembly PCR

Answer 8

ya

Question 9

what is the purpose of random mut?

Answer 9

–analyze the important of specific aas when not much is known about the protein

Question 10

ways to introduce random muts?

Answer 10

– Chemical mutagenesis
• Expose DNA to chemical mutagens
– Error-prone PCR & misincorporation
• PCR reaction with error-prone polymerase
• Limiting concentrations of one dNTP in the presence of dITP (I = Inosine)
• Presence of Mn2+
-gene shuffling
-directed evolution–multiple rounds of mut and selection for improved prot function

Question 11

what is PCR-based RM?

Answer 11

take ds PCR products with errors at random positions–>ligate into vector–>clone and express–>select for a particular phenotype

Question 12

quikchange–had mutated oligo primer, extend, use ds/ss, linear or circularized DNA

Answer 12

ya

Question 13

QuikChange mutagenesis kit
– two mutagenic primers are complementary to each other
– 25-45 bases in length
– 10-15 bases on each side of mutation(s)
– GC content of 40% - 60%

Answer 13

ya

Question 14

read slide 24

Answer 14

ya