MetaMorph Mutagenesis Kit

Overview:

metamorph

Simple, Rapid, and Highly Efficient PCR-Based Mutagenesis Kit

MetaMorph is a mutagenesis kit based on mutagenic PCR fragments that are generated with a high-fidelity DNA polymerase, custom-designed mutagenic primers and vector primers. The mutagenic primers are designed to incorporate single or multiple mutations, insertions, or deletions. Vector primers are designed to have 20 bp homology to the linearized vector ends. The mutagenic PCR fragments fuse to create the final mutated gene-of-interest. The kit is so robust that multiple mutagenic DNA fragments can be assembled simultaneously and cloned into one construct in a single step. MetaMorph is more than 90% efficient, without unwanted mutations being introduced.

Technical Details:

metamorph details

Forward and Reverse Vector Primer Design
The forward vector primer (in red) should be generated to the positive strand of DNA, and should contain ~20 bases corresponding to the vector sequence leading up to the restriction site where the gene-of-interest has been inserted. The reverse vector primer (in blue) should be generated to the negative strand of DNA, and should also contain ~20 bases complimentary to the vector sequence leading up to the restriction site where the gene-of-interest has been inserted.

Mutagenic Primer Design
The reverse mutagenic primer (in red) should be generated to the negative strand of DNA, and should contain 10 bases 5’ of the mutation, the mutagenic bases, and then 20 bases 3’ of the mutation site. The forward mutagenic primer (in blue) should be generated to the positive strand of DNA, and should contain 10 bases 5’ of the mutation, the mutagenic or inserted bases, and then 20 bases 3’ of the mutation site. The forward and reverse mutagenic primers, therefore have a region of ~20 bases that is entirely complimentary and that contains the mutagenic bases.