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Flow chart of the single-primer site-directed mutagenesis method. The parental plasmid is shown in grey color and the two PCR synthesized strands are shown in blue and purple. The letter marks the position of the mutation.
Procedimento num nico tubo de reaco ( 2 primers complementares por tubo de reaco)
Mutagenesis
Mutagenesis -> change in DNA sequence -> Point mutations or large modifications? Point mutations (directed mutagenesis): - Substitution: change of one nucleotide (i.e. A-> C) - Insertion: gaining one additional nucleotide - Deletion: loss of one nucleotide
Consequences of point mutations within a coding sequence (gene) for the protein
Silent mutations: -> change in nucleotide sequence with no consequences for protein sequence
SiteDirectedMutagenesis
Thereactionwasperformedusing PfuTurbo DNApolymeraseandtwosynthetic oligonucleotideprimerscomprisingthe desiredmutation.
SiteDirectedMutagenesis
Oligonucleotideprimers(oligos)were designedtoincorporatethedesired mutation,andthePfuTurbo DNApolymerase replicatesboththeplasmidstrandswithhigh fidelitywithoutdisplacingthemutantoligos
SiteDirectedMutagenesis
Followingthethermalcycling,theproduct wastreatedwithDpn Ienzyme(target sequence5Gm6ATC3).
SiteDirectedMutagenesis
ThenickedvectorDNAcontainingthe mutationwasthentransformedintoXL1Blue supercompetent cells.
Oligonucleotideprimerdesigning
Inmostcases,theprimersweredesignedin suchamannersoastofacilitateeither incorporationofanewREsiteorremovethe existingREsite.
Oligonucleotideprimerdesigning
Thiswasachievedbycreatingsilent mutationsinoraroundthedesiredmutation region.
Oligonucleotideprimerdesigning
Boththeforward andreverse primers containedthedesiredmutant.
Oligonucleotideprimerdesigning
Theprimersannealtothesamesequenceon oppositestrandsoftheplasmid(schematically showninfigure).
Oligonucleotideprimerdesigning
Thelengthoftheprimerswassetto approximately25 30bases. Carewastakentohaveaminimumof10 bases flankingthemutantsite. GCcontentwasmaintainedbetween40 60 %.
Oligonucleotideprimerdesigning
Thelengthoftheprimerswassetto approximately25 30bases. Carewastakentohaveaminimumof10 bases flankingthemutantsite. GCcontentwasmaintainedbetween40 60 %.
Questes
1 quaissoasprincipaisdiferenasdeSDM (sitedirectedmutagenesis)baseadaemPCRem relaoaumareacostandarddePCR? 2 quandoomoldedareacoumplasmdio, podedizersequeoprodutodareaco tambmumplasmdiocircular,intactoe superenrolado? 3 Porquesedizqueoplasmdiomolde biosintetizado(poroposioasintetizadoin vitro)?
QuecaractersaticasdaenzimaDpnIsorelevantes paraestetrabalho?