Lecture 3/4 Applied Molecular Biology Mutagenesis - mutants are used to study the roles of individual regions or AAs within a protein Traditional methods: Random mutagenesis of whole genome, select for mutants which lack the activity of interest Random mutagenesis by PCR: Random mutagenesis of the gene or region of interest only Newer methods (site specific) - Cassette mutagenesis - Site directed mutagenesis - Combinatoral mutagenesis (High throughput) - Molecular evolution (High throughput) Traditional · Treat organism with mutagen e.g. UV or chemical mutagen · Accept high kill rate up to 95% · Screen survivors for clone with loss of activity of interest . No idea how many mutations or where they are located Random by PCR · Use error prone polymerase (no 3' to 5' exonuclease) can't cut out mistakes and no proofreading activity · Use high conc on dNTPs · Use Mn2+ rather than Mg2+ . All these encourage mistakes to be made which are then amplified in subsequent rounds of PCR . The mutations are now all located in the gene of interest, which must now be clones and expressed Cassette RE1 Play *RE2 Digest, purify plasmid & RE1 * RE2 (remove insert) Add new insert and ligate RE1 ~ RE2 NB. New insert can be: Synthetic DNA A digested PCR product (A fragment of a DNA digest) But MUST have complementary ends for the ligation.
Site Directed Mutagenesis - MegaPrimer PCR The product after both rounds is a DNA cassette (has right lengths and has mutations in both strands), used in cassette mutageneis with the original gene. Round 1: primer 1 and Mutant primer to form the mega primer Round 2: mega primer and primer 2 used. Only 1 strand of mega primer used Leads to both strands of the cassette having the mutation While only 1 strand is used the other may anneal to the complementary stand of DNA however some Taqs have 5' to 3' exonuclease and will chop it up, others will just displace it. 5 1 3' RE1 5 M 25' PCR with 1 + M RE2 3 5' 5' 5 3 3' 3 2 5 PCR with megaprimer + 2 5ª 3 3 5 Megaprimer (must be purified) '5' 3 3 5' Play Candidate No: 5 3' vis' 5 VIS' 3 3 5 3' 3' 5 3 Ky 3' 5' 2 primer mutagenesis (quick change) - quicker but technically harder Cycle 1 · You have 2 primers, each containing mutation of interest · Requires high fidelity polymerase (one that doesn't make mistakes) Cycle 2
. Polymerase stops when it reaches the primer, important it doesn't have strand displacement activity otherwise It would go 5' 3' round and round. Play 3'5' High fidelity polymerase 3 · First cycle gives you hybrid plasmid Cycle 1 · Second cycle give you hybrid plasmid and plasmid both strands with mutation New DNA 3'5'A 5'3' · Once double stranded product made can't copy anymore because of gaps in strand making it linear. This means poor yield