Mutagnese stio dirigida

Site Directed Mutagenesis

Procedimento em duas reaces separadas (1 primer por tubo de reaco)

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)

Directed Mutagenesis and Protein Engineering

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

-> Change of amino acid -> truncation of protein

-> change of c-terminal part of protein

-> change of c-terminal part of protein

Applications of directed mutagenesis

Produo de protenas mutadas

Protenas com interesse em Biotecnologia podem ser alteradas por mutagnese O gene mutado clonado num organismo (por exemplo E. coli) e a protena recombinante mutada produzida em larga escala para uso na indstria

O Sistema QuikChange Site-Directed Mutagenesis Kit (Stratagene)

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 %.

 Theoligonucleotidesequencewasfurther analysed foranyhairpinformationordimmer formations usingonlinesoftwarefromNet PrimerofPremierbiosoft orbyusingJellyfish 1.5fromBiowire.

Questes
1 quaissoasprincipaisdiferenasdeSDM (sitedirectedmutagenesis)baseadaemPCRem relaoaumareacostandarddePCR? 2 quandoomoldedareacoumplasmdio, podedizersequeoprodutodareaco tambmumplasmdiocircular,intactoe superenrolado? 3 Porquesedizqueoplasmdiomolde biosintetizado(poroposioasintetizadoin vitro)?

 4 Qualaestratgiaparaeliminaroplasmdio moldeoriginalapsareacodeamplificao? 5 Especificamenteparaoobjectivodapergunta anterior,quecaractersticagenticadevepossuir aestirpedeE.coliutilizada? 6 Consultando

http://products.invitrogen.com/ivgn/product/15242019?ICID==%3Dsearch15242019 Ou http://www.neb.com/nebecomm/products/productr0176.asp

QuecaractersaticasdaenzimaDpnIsorelevantes paraestetrabalho?