Thursday, April 17, 2014

Design a mutagenic primer to engineer human C333F mutation to yeast MSH2

Goal: Engineer the human C333F mutation into yeast MSH2.

Link of video tutorial of this exercise:

Video on G548C is

The principle of site specific mutagenesis can be found in this tutorial video. Explanation of cognant sites can be see in this Youtube video.

Copy yeast and human MSH2 protein sequences in FASTA format from this link. 

Google "EMBL ClustalW2".  Then, paste the yeast and human MSH2 sequences in FASTA format into ClustalW2 window.

  After "submit", you should see the protein alignment.

Identify the cognant site in yeast MSH2. Human C333 corresponds to yeast C345.

Therefore, human mutation C333F should become C345F mutation in yeast MSH2 gene.

Now, we can design the mutagenic primer based on the DNA coding sequences of the yeast MSH2 (linked here).  You can copy-paste this sequences into ApE.

In the yeast MSH2 ORF sequence, the 345th amino acid position corresponds to 345x3=1035 nucleotide position. So, the codon position is 1033-1035.  We can select these 3 nucleotide in ApE to and double check this codon.

This selected codon should be:
We can verify that  codon TGC is amino acid "C".

To design a mutagenic primer, we will pick 10 bp left to the TGC codon and 10 bp right to the TGC codon, by select from 1023-1045.

Copy the selected sequence into a new ApE window.

At the center of this sequence should be "TGC".

The yeast codon usage table is:

The preferred codon for "F" in yeast is "TTT". 

We can manually change "TGC" to "TTT".

We can save this file in ApE as "C345F_mutagenic_primer.seq".

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