A Single Step Procedure to Mutagenize Multiple Sites of a Gene
|Affiliation: Institute of Translational Pharmacology, National Research Council, Rome, Italy|
|Source: Protocol Online|
|Date Added: Mon Feb 18 2013|
|Date Modified: Wed Feb 20 2013|
|Abstract: A single step method to obtain multiple mutations.A modification of Quickchange site-specific mutagenesis protocol.|
This technique is a modification of the protocol of commercial site-directed
mutagenesis kits and relies on a simultaneous annealing of specific mutagenic
oligonucleotides on different sites of the template, to obtain in a single
reaction single and/or multiple mutations (1,2). We have used this strategy to
introduce two amino acid substitutions, S46A and S64A, in the human Tumor
Protein Translationally-Controlled 1 gene (TPT1)[EMBL:BC040008.1].
The two couples of complementary oligonucleotides used to mutagenize cDNA,
were obtained from web-based program PrimerX (bioinformatics.org/primerx).
Each pair of oligonucleotides is about 40 bp long and contains the specific substitution in the middle of the sequence.
The Stratagene Quickchange site-directed mutagenesis protocol provides a
description of the method and is available as a PDF file at: (chem.agilent.com/Library/usermanuals/Public/210518.pdf).
95°, 2’(1 cycle); 18 cycles: (95°, 20”; 60°,10”; 68°,2 minutes/kb of plasmid length) ( 68°, 5’), one final cycle.
After PCR amplification, add 1 µl of DpnI
restriction enzyme to half of the reaction and incubate for 2 hr at 37°. Use 12
µl of the digested product to transform DH5α
chemically competent E.coli.(3). Sequence 5-10 of the resulting colonies.
By sequencing 5 colonies of 57 obtained, 40% of colonies were double mutated,
60% were mutated in only one site (among these 20% were mutated also in