#SiteDirectedMutageneis is most widely achieved by the two-primer #PCR or the #Quikchange approach (as per #Agilent's kit named Quikchange) which is achievable for #insertion, #deletion as well as #substitution mutations. It is one of the simplest ways to achieve it and has fairly standardized kits and protocols available in the market.
Principle
The principle of Quikchange SDM solely depends on fairly long #megaprimers which are often 30-35 bases long with the intended mutation-bearing bases in the middle of it. #PCR is performed with a high-fidelity #DNApolymerase for ~18 cycles and a bit lower extension temperature (often 68C). With the rounds of PCR, the mutation present in the primer is incorporated in the product plasmids. The #methylated and #hemimethylated parent plasmids are get rid of with #DpnI treatment. After that the PCR product is transformed in preferably #HtePhenotype E. coli #CompetentCells. The nicks which are present at the 3' position of the plasmid strands, are sealed inside the cells with help from the intrinsic #DNALigase.
Protocol
a. Primer design: the primers should have two or all of the following:
30-35 bases in length
mutation in the middle, or at least having 10 bases 3' of the mutation
Tm of 75-85C
b. PCR with high-fidelity #DNAPolymerase, for ~18 cycles and a bit lower extension temperature (often 68C). #DMSO is a common additive for >4kb templates.
c. #DpnI treatment of the PCR product.
d. Transformation in #HtePhenotype E. coli #CompetentCells.
e. #Clone selection by means of #ColonySequencing and/or #ColonyPCR.
Merits
Efficiency of at least 80%
Possible to mutate upto14kb of DNA templates.
Demerits
Costlier than other alternatives.
Troubleshooting tips: https://www.biobulletins.com/post/improve-site-directed-mutagenesis-results
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