gene cloning and amplification

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roniadam
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gene cloning and amplification

Post by roniadam » Wed Apr 23, 2008 8:52 am

Hi everyone:

I am confused with gene cloning and Gene amplification. Is there a relationship between them?
Why do we have to clone the gene if we can amplify the gene fragment? Is it necessary to amplify the gene after clning for expression?

blcr11
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Re: gene cloning and amplification

Post by blcr11 » Wed Apr 23, 2008 1:53 pm

The "concentration" of specific gene sequence in a sample of genomic DNA is quite small. Rarely would there be enough raw DNA to effect a cloning. So you amplify the specific sequence you want so that it becomes the overwhelmingly dominant sequence present. Then more than 99% of the DNA in the sample is the gene you want to clone, and you do so. You amplify in order to clone (usually).

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Post by blcr11 » Wed Apr 23, 2008 2:01 pm

Sorry, the other half of the question: You don't need to amplify the gene sequence after you've cloned it in order to express it. You clone the gene into a construct that has whatever regulatory sequences you need depending on where you intend to express the gene (promoters, TATA box, stop codon, for E coli; enhancers sometimes for mammalian cell expression; insect-specific upstream or downstream elements for baculovirus expression etc). You may induce expression especially when using E coli, but that's not amplification of the sequence. Induction is turning on gene expression by removing a repressor most typically, though there are probably other ways to induce.

roniadam
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Post by roniadam » Wed Apr 23, 2008 3:50 pm

Thank you very much. very good and convensing answer.

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