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Gene of interest isolation
#1
Can you please explain the following in a simple language- When we isolate nucleic acid from any tissue like cheek cells and go for restriction endonuclease treatment, we get huge numbers of fragments. These fragments represent portions of DNA from different chromosomes. Now if we do electrophoresis of the same, we will gey bands at different distances, based on sizes. Now I have three questions- 01. How we identify and isolate the gene of interest from here. 02.How we allocate the fragments containing genes to respective chromosomes. 03. For long genes, how do we ascertain that the entire gene is represented by restriction endonuclease treatment. Thanks and regards.
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#2
(04-29-2015, 06:08 PM)S Ghosh Wrote: Can you please explain the following in a simple language- When we isolate nucleic acid from any tissue like cheek cells and go for restriction endonuclease treatment, we get huge numbers of fragments. These fragments represent portions of DNA from different chromosomes. Now if we do electrophoresis of the same, we will gey bands at different distances, based on sizes. Now I have three questions- 01. How we identify and isolate the gene of interest from here. 02.How we allocate the fragments containing genes to respective chromosomes. 03. For long genes, how do we ascertain that the entire gene is represented by restriction endonuclease treatment.  Thanks and regards.
Hello S Ghosh,

In my understanding, (i) southern blotting should help in finding the band in which gene of interest is there (ii) For a random digestion using same RE for the same pool of DNA (cheek cells), finding out the chromosomal affiliation of the gene is very difficult (not possible in my view). (iii) a judicial choice of restriction endonuclease whose cutting regions are there in the flanks of the gene only, will ensure that you get the entire gene (websites like neb-cutter can help in predicting the fate of your gene of interest by using different restriction enzymes, if you know the sequence of your gene/chromosome).

I would be interested in knowing the answer to your second question as well.

Rajni
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#3
Thanks Rajni. But I have the feeling that we always presume that there will be RE regions flanking each gene. Is it a Universal feature. If not then how can one ascertain the particular gene, even by using probe.
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