Saturday, 17 April 2010

molecular biology - What is the least costly method to generate sequential amino acid deletions?

Deletions may make sense if you are analyzing the N-terminus or C-terminus of a protein. If you are looking at an internal region however, keep in mind that the more AAs you delete, the more likely you are to disrupt the overall protein structure. If you delete any random selection of 8 AAs within a protein, there's a chance you'll knock out activity by changing the protein fold or stability. That's not useful information.



This question is usually first addressed by alanine scanning - sequentially or additively changing amino acids to alanine. This is much more informative than deletions. Even better, you can choose to replace wild-type AAs with other AAs of similar size but differing charge or hydrophobicity. Then you are most likely to change the function of a region without changing the structure.



The quickchange kit works great, but if cost is an issue you can do whole plasmid mutagenesis PCR with your own reagents. And make your own competent cells.



In my experience though, whole plasmid PCR can be tricky - if it doesn't work the first time it can be difficult to troubleshoot. If time is an issue, I'd recommend doing overlap extension PCR with the same mutagenic primers, plus one set of amplification primers at the 5' and 3' end of your gene. Make a large batch of digested vector, test as a negative control, and use it for ligation of all of your different inserts.

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