Top : Molecular Biology : DNA : Mutagenesis : PCR Mutagenesis : A Single Step Procedure to Mutagenize Multiple Sites of a Gene

A Single Step Procedure to Mutagenize Multiple Sites of a Gene

Author: Stefania.Ciafre
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).

Procedures

PCR assembly

PCR conditions

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 l of DpnI restriction enzyme to half of the reaction and incubate for 2 hr at 37. Use 12 l of the digested product to transform DH5α chemically competent E.coli.(3). Sequence 5-10 of the resulting colonies.

Results

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 unspecific sites).

References

  1. Liu H, Naismith JH. (2008) - BMC Biotechnology 8(1):91. PMID:19055817
  2. Kirsch RD and Joly E. (1998) - Nucleic Acids Research 26:1848-1850. PMID:9512562
  3. Chung,C.T and Miller,R.H. (1988) - Nucleic Acids Res. 16(8),3580. PMID:3287331
Printer friendly page