RACE vs. cDNA screening

Warren Gallin wgallin at gpu.srv.ualberta.ca
Thu Jul 12 11:24:31 EST 2001


Hi,
	We are doing exactly this, and have had great success with the
Invitrogen Generacer kit (no affiliation), but there are other kits that
are supposed to do the same thing.  We directly sequence the PCR
products to avoid sequence errors from cloning a mistake.  The advantage
from my point of view is that we are after a few sequences from a large
number of species, and making 10 libraries is not cost-effective or time
effective for us.  If you are planning on doing a lot of work on your
particular tissue/organism then making the library might be a better
decision, logistically.
	As far as the issue of isoforms, you need to isolate the 5' ends and 3'
ends separately by RACE and sequence them, then design a new set of
primers from the 5' end and 3' end and do a second round of simple
RT-PCR to identify the real full-length transcripts.

Warren Gallin

Ricky Boernke wrote:
> 
> Dear all,
> 
> using degenerate primers I was able to PCR an internal portion of
> my gene of interest. Since I am also interested in the 5' and 3'
> regions, respectively, I was thinking about the appropriate method
> to identify these parts of the cDNA too. I could easily use my PCR
> fragment as a probe to screen a respective cDNA library, however,
> this is quite time and labour consuming. Thus, I was also
> considering to run a RACE PCR. But what you here from other
> people is that doing RACE is often problematic.
> Moreover, I expect my gene to be represented
> by several isoforms which I think could become a problem when
> assembling the RACE fragments into a contigous cDNA. You can never
> be sure whether you glue the proper 3 and 5' ends together and thus
> might end up with a chimeric clone. Or wouldn't you regard this as
> a problem. I don't now yet how different the isoforms will be.
> Any input welcome
> 
> Cheers
> Ricky




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