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For protein production, the gene of interest is inserted in an expression vector and introduced into a suitable host for expression. But how is the gene of interest for the desired protein isolated from the organism which possesses it?

One way could be to synthetically make the DNA sequence coding for that protein. Another could be to identify the gene of interest from a pre-existing genomic library or cDNA library of that organism. Are they the only methods?

The organism's entire genome could be taken and cleaved with restriction endonucleases, and a Southern blotting technique (i.e. electrophoresis followed by probe hybridisation and autoradiography) could be used to screen the gene of interest from the fragments produced. Of course, for this the gene sequence must be known beforehand, in order to synthesise the probe. Is this method also adopted? It seems inefficient - given that the fragments are assumed to have the intact gene of interest, and the gene should have been sequenced beforehand.

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As you already wrote there are two basic ideas: Synthesis of the desired gene sequence and use of existing DNA.

DNA Synthesis is getting cheaper so it is becoming the method of choice for generating DNA-samples up to about 5000 bp (for example see the IDT-page for commercial gene synthesis). If this is too short for your desired sequence there is the possibility to split the gene to different of those synthetic sequences and to fuse them using Gibson-Cloning (Gibson et al. 2009) - after adjusting the synthetic sequences so that there is an overlapping sequence. Alternatively you could use classical cloning - provided there are suitable cleaving sites in your sequence.

If you don't want to synthesize the DNA and have a template available you could design suitable primers and amplify the gene from the template using PCR and purify it by gel-electrophoresis. Inserting the sequence into a vector then requires that you find suitable cleaving sites within the sequence and the vector.

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  • $\begingroup$ Using existing DNA as in using it from a genomic library or by isolating gene of interest from the organism's genome by the method described in the question? $\endgroup$ – Charles May 13 '16 at 5:10
  • $\begingroup$ You could either use a genomic library or you could isolate genomic DNA from the organism. In both cases you wouldn't have to digest it as described in the question, but you could amplify the gene of interest directly from a low amount of DNA (NEB state 10000 copies of DNA are necessary for sufficient amplification in 25-30 cycles). $\endgroup$ – aretaon May 13 '16 at 11:26

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