Mutagenesis using DpnI selection

목차

1. Introduction
2. Design of primers
3. Thermostable DNA polymerase
4. Materials
5. Method
6. References

본문내용

Introduction
In vitro mutagenesis using double-stranded DNA templates: selection of mutants with DpnI allows desired mutation in double stranded plasmid.
The entire lengths of both strands of the plasmid DNA are amplified by primers containing the desired mutation in PCR. The because DpnI endonuclease specifically cleaves fully methylated GMe6ATC sequence. DpnI is used to digest the parental DNA templateand to select for mutation-containing synthesized DNA. The desired mutants are recovered by transforming E. coli to antibiotic resistant.

Step 1 Step 2 Step 3 Step 4 Plasmid Preparation Temperature Cycling Digestion Transformation
fig 1. overview In vitro mutagenesis with DpnI

Design of primers
- Must anneal to the complementary strands of the same target sequence.
- Must be of equal length (between 25 and 45 bp), with a melting temperature of 78°C or greater.
- The desired mutation should be in the middle of the primer with ~10-15 bases of correct sequence on both sides.
- The primers optimally should have a minimum GC content of 40% and should terminate in a G or C residue.

참고 자료

Sambrook and Russell. 2001. Molecular cloning 3rd edition New York: Cold Spring Harbor Laboratory vol 2: 13.19-13.25.
QuikchangeⓇ Site-Directed Mutagenesis Kit. Instruction Manual. StratageneⓇ