Alternative to RACE-PCR

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magicsiew
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Alternative to RACE-PCR

Post by magicsiew » Sat Jan 15, 2011 3:59 pm

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...

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JackBean
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Post by JackBean » Sun Jan 16, 2011 9:52 am

what about some genomic or cDNA library?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Sun Jan 16, 2011 10:10 am

Able to find partial sequence in the EST only

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JackBean
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Post by JackBean » Sun Jan 16, 2011 11:15 am

use it as probe for search in DNA library and from that you should be able to find larger piece of your gene.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Sun Jan 16, 2011 3:00 pm

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

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JackBean
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Post by JackBean » Sun Jan 16, 2011 3:11 pm

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...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Sun Jan 16, 2011 4:52 pm

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...

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JackBean
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Post by JackBean » Sun Jan 16, 2011 5:59 pm

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.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Sun Jan 16, 2011 6:18 pm

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

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JackBean
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Post by JackBean » Mon Jan 17, 2011 9:27 am

which second method?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Mon Jan 17, 2011 9:42 am

Hybridization...

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JackBean
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Post by JackBean » Tue Jan 18, 2011 6:49 am

but that's what I was talking about all the time ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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