Alternative to RACE-PCR

[magicsiew]

Hi, to clone out full length of a gene from a genome without complete sequencing, the way I know can use is perform RACE-PCR. However, RACE-PCR very expensive. Besides this method, is there any other method to clone out the full length of gene?

One method I have thought about is designed another primers set that are able to amplify the full length of the gene, but in my case, the primers are degenerate and were designed from conserved region from few species. Without the complete genome sequence, I think this method dont really work rite?

Hope to get some other idea from here. Thanks...

[JackBean]

what about some genomic or cDNA library?

[magicsiew]

Able to find partial sequence in the EST only

[JackBean]

use it as probe for search in DNA library and from that you should be able to find larger piece of your gene.

[magicsiew]

But there is no DNA library yet... At least now still dont have...

[JackBean]

then make some That's not that hard. You just need to isolate either gDNA or mRNA (depends on what type of library you wanna make) and pick some appropriate vector...

[magicsiew]

Hmm... I am not sure if I get your meaning right? Are you saying making DNA library, sequence them, then use the fragment as probe, align them and spot the region for full length of the gene? If this is what you meant, will it be more expensive than RACE-PCR? Say if I only need to carry out 4 RACE-PCR...

[JackBean]

Well, I thought you have some problems with the RACE-PCR and that's way you're looking for other opportunities.

Anyway, what I meant is: if you have part of your gene in database. You can synthesize such a piece of DNA (e.g. by service provided by Mr.Gene).
Separately, produce DNA library, either from gDNA or mRNA. And use this library to trace your gene by hybridization.

[magicsiew]

I think I will go for the second method.. Thanks

[JackBean]

which second method?

[magicsiew]

Hybridization...

[JackBean]

but that's what I was talking about all the time