gene cloning and amplification

[roniadam]

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]

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).

[blcr11]

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]

Thank you very much. very good and convensing answer.