how to construct the gene of a functional domain? - (Jun/13/2007 )

I'm preparing a proposal (required for an exam). And the expression of a certain protein domain was not described in details. In the paper, they mentioned “The gene for MMP-2C was constructed by PCR amplification using human full-length MMP-2 as the template ”

I don't know about gene construction. I found out the amino acid sequence of this domain. But I really have no idea about the following. Would you guys please give me some hints? Reference books are also good.

Thanks a lot.

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If I'm understanding correctly, it sounds as though they are trying to express the functional domain of MMP-2. Therefore, they used PCR to amplify the region they wanted. That means, they found the sequence of this gene (similar to how you got the amino acid sequence), designed primers with restriction sites which would anneal at either end, and performed PCR. This gave them an insert, which they could put in an expression vector and transform into bacteria to produce their protein (or portion of protein) of interest.

Does that help?


Amanda

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if they did not reference the primers and you need them, you may be able to get them if you email the authors? how much detail do you need in your proposal?

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