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How to clone a gene whose genome has not been sequenced yet?

ortholog cloning

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#1 IndiaNita

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Posted 12 April 2014 - 02:41 PM

Hi all,

How to clone a gene whose genom has not been sequenced yet?
Size of an unknown gene is 5kb.
Unknown gene has high homology with ortholog gene, and we know the sequence of ortholog gene.

I don't know initial steps.
I know what to do when I get the sequence of the unknow gene.
Is anyone have any idea?

All comments and answers are welcome smile.gif 

Thanks in advance!



#2 phage434

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Posted 12 April 2014 - 07:16 PM

The normal approach is to find regions of the protein that are highly conserved, and that have codons with limited variability. Single codon AAs such as methionine or tryptophan are useful since you know with certainty their nucleotide representation. Try to design primers for a short segment of your gene using these conserved known regions, along with degenerate bases in positions that are uncertain. If you are working with a eukaryote, then you probably should extract RNA and do an RT reaction so that you are working without introns.

 

When you have amplified a small section of the gene, then you can sequence it. Once you have sequence, you can make outward directed primers, and do an inverse PCR reaction to obtain adjacent nucleotide sequence, and work your way out by repeating as necessary.



#3 IndiaNita

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Posted 13 April 2014 - 02:43 AM

I found this in one article, about Southern blotting : " Alternatively,if the gene has been cloned from a related organism,one in which the gene sequence is likely to be similar to that in the organism now being studied, then heterologous probing can be used,in which some noncomplementarity between probe and target is tolerated during the hybridization step. This approach can be used, for example,to identify homologues of human genes in the genomes of other primates,and can even be used across broad species barriers,for example by using Drosophila genes as probes for related human sequences."

As I understood, I can use a sequence of ortholog gene as a probe, to find unknown sequence?

And when I do, what is the next step?

Actually, what is the step between Southern blot and inserting sequence in plasmid?
 



#4 phage434

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Posted 13 April 2014 - 05:02 AM

There are some techniques for cloning fragments of the genome "blind." You can do this with the entire genome, or use Southern blotting as you describe to locate randomly cut fragments containing the gene on a gel, then clone from DNA  fragments in the gel at that position. After cloning, you have to do colony blots to identify clones which also have the correct insert. I'd judge these methods more difficult and time consuming than the one I outlined, but both are rather challenging.



#5 IndiaNita

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Posted 14 April 2014 - 12:01 PM

yes they are, but also much more safer for me, since i'm not that handy in lab smile.png  yet rolleyes.gif 
thank you very much!







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