Cloning a novel sequence.

[KAZU]

I'm new here and I have no idea if this is the correct forum to post this thread on or not, but here it is.

 

The first question: How do I clone a novel gene that encodes a protein that has sufficient evidence to support its existence?

 

Since I don’t want to screen libraries, my first thought is this:

 

1) Compile sequence alignment blocks of known homologous proteins.

2) BLAST against whole genome of chosen animal or trace archive.

3) Build a contig and use it to design GSPs.

4) Test the GSPs with genomic template for animal of choice.

5) Go into 5’ and 3’ RACE.

 

The second question: If I decide to go with my first thought, how do I do parts 1 and 2? I suck at computers.

[CharonY]

Actually if you already have homologous sequences (as your approach suggests) then you could simply align those sequences and look for conserved regions, make primers against them, blast those primers against databases to check if they are specific for this gene and try to amplify it.

If you want to see if it is expressed a simple RT-PCR should suffice.

[KAZU]

Charon, the problem with that is there is no way to know if it is specific for the gene I am interested in since the gene I'm interested in might not exsist in any database......yet. If it does exisist it would only be in a trace archive packed full of introns since not all animals have its genome sequenced. So I would need a good chunk of sequence.

[zyncod]

I don't understand how you plan on building a contig if you're not going to screen libraries to some extent. Also, once you have GSPs, why would you do RACE? Are you interested in the regulatory regions?

 

If I was doing this, the first thing that I would try - after BLASTing, of course - would be a Southern for homology (which, assuming a positive result, would be followed by library screening, followed by further Southerns to isolate the region in the cosmid/plasmid/etc, followed by sequencing and computer analysis).

 

Edit: Sorry. Given that I don't know what you mean by 'contig' in this context, it is entirely possible that your GSPs do not fully encompass the gene of interest. In which case RACE would be entirely appropriate. It would really help if you indicated what type of gene in which organism you are looking for.

[KAZU]

Sorry, to clearify my personal definition of building a contig. In this case I define it as exctracting partial sequence from a data base and then blasting it against the genome of a specific organism. Then analyze the output sequence alignment by blasting sequences that partially align with the original sequence against the entire data base to check for homology then manually aligning the sequence overlaps to create a more complete sequence.

[zyncod]

Well, that would imply that your organism has been sequenced. If not, your GSPs from another organism are almost guaranteed not to work. It's worth a try, but I think you might also want to try the southern approach, as a positive result would guarantee that you could eventually isolate your gene.

[KAZU]

My organism is not fully sequenced, there are partial sequences available in the trace archives. I will also look into the hybridization techniques, I just don't want to go there, I'm way too impatient.