Exercício Mutagénese sítio dirigida - questões.pdf
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Transcript of Exercício Mutagénese sítio dirigida - questões.pdf
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Mutagnese stio dirigida
Site Directed Mutagenesis
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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.
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Procedimento num nico tubo de reaco
( 2 primers complementares por tubo de
reaco)
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Directed Mutagenesis
andProtein Engineering
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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
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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
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Applications of directed mutagenesis
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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
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O Sistema QuikChange Site Directed Mutagenesis Kit (Stratagene)
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O Sistema QuikChange Site-Directed Mutagenesis Kit (Stratagene)
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SiteDirectedMutagenesis Thereactionwasperformedusing
PfuTurboDNApolymerase
and
two
synthetic
oligonucleotideprimerscomprisingthe
desiredmutation.
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SiteDirectedMutagenesis Oligonucleotideprimers(oligos)were
designedto
incorporatethedesired
mutation,andthePfuTurboDNApolymerase
replicatesboththeplasmidstrandswithhigh
fidelitywithout
displacing
the
mutant
oligos
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SiteDirectedMutagenesis Followingthethermalcycling,theproduct
wastreated
with
DpnIenzyme(targetsequence5Gm6ATC3).
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SiteDirectedMutagenesis ThenickedvectorDNAcontainingthemutationwasthentransformedintoXL1Bluesupercompetent cells.
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Oligonucleotideprimerdesigning Inmostcases,theprimersweredesignedin
suchamanner
so
as
to
facilitate
either
incorporationofanewREsiteorremovetheexistingREsite.
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Oligonucleotideprimerdesigning Thiswasachievedbycreatingsilentmutationsinoraroundthedesiredmutationregion.
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Oligonucleotideprimer
designing
Boththeforwardandreverseprimers
containedthe
desired
mutant.
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Oligonucleotideprimer
designing
Theprimersannealtothesamesequenceonopposite
strands
of
the
plasmid
(schematically
showninfigure).
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Oligonucleotideprimer
designing
Thelengthoftheprimerswassetto
approximately25
30
bases.
Carewastakentohaveaminimumof10basesflankingthemutantsite.
GCcontentwasmaintainedbetween40 60
%.
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Oligonucleotideprimer
designing
Thelengthoftheprimerswassetto
approximately25
30
bases.
Carewastakentohaveaminimumof10basesflankingthemutantsite.
GCcontentwasmaintainedbetween40 60
%.
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Theoligonucleotidesequencewasfurther
analysed forany
hairpinformationordimmer
formationsusingonlinesoftwarefromNet
PrimerofPremierbiosoft orbyusingJellyfish
1.5from
Biowire.
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Questes 1 quaissoasprincipaisdiferenasdeSDM
(site
directed
mutagenesis)
baseada
em
PCR
em
relaoaumareacostandarddePCR?
2 quandoomoldedareacoumplasmdio,
pode
dizer
se
que
o
produto
da
reaco
tambmumplasmdiocircular,intactoesuperenrolado?
3
Porque
se
diz
que
o
plasmdio
molde
biosintetizado(poroposioasintetizadoinvitro)?
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4Qualaestratgiaparaeliminaroplasmdiomoldeoriginalapsareacodeamplificao?
5 Especificamenteparaoobjectivodaperguntaanterior,quecaractersticagenticadevepossuiraestirpedeE.coliutilizada?
6 Consultando
http://products.invitrogen.com/ivgn/product/15242019?ICID==%3Dsearch15242019
Ou
http://www.neb.com/nebecomm/products/productr0176.asp
Quecaractersaticas
da
enzima
DpnI
so
relevantes
paraestetrabalho?
