question in 5' RACE and primer extesion - (Apr/17/2006 )

I want to check a gene's mRNA ends recently. For 5' end, there are commonly two options as you know, 5' RACE and primer extension. I want to use 5' RACE to figure it out, however, primer extension is the best method. I am not sure if 5' RACE is really good enough to get the result. Also even if yes, I do not know what company has the best kit for this method. Can anyone tell me if you had good expereince on this kind of kit? Thank you very much.

six

Please help.

Sequencing a 5' RACE pcr product should identify the 5' end of a transcript reliably if it is a single product. If there are multiple initiation sites, you probably would have to clone the product into a vector and sequence a dozen or so to get good statistics. You could tell if this was necessary by sequencing from the 5' RACE primer, which would give mixed sequence if there were more than one start site.
Why do you think you need a kit? Superscript III cDNA production, ligation of a 5' RACE primer with T4 RNA ligase, and PCR should do the trick.

No preference, but I use Clontech's since my lab has one. The trick is designing the primers and preferentially, do a nested PCR. If it's a multicopy gene family, then you'll have to do cloning as mentioned by phage434.

Phage434 and Isek, Thanks a lot for your suggestions. Then I am planning to do that by using a clontech kit since we have two in the lab. becuase i do not know if my target gene has multiple transcription start point in its mRNA, I may have to do TA clone and sequence many inserts, is it better to choose longest inserts for sequencing firstly? Also, it is really trick to design the primers because the organism i am working with has very high A/T rich sequence in the whole genome. I have to desgin sometimes more than 40 bps for one primer to get the high Tm, but the G/C% is still low.
six

For low GC organisms like this, you should be aware that it is often necessary to use a low extension temperature (64-66C) rather than the normal 68 or 72 C. Primers longer than about 30-35 bp will still denature at high temperature, even if the predicted Tm is higher (the predictions are wrong). You should just plan on a low annealing temperature. Try to make the 3' end of the primer have at least some GC content -- end in a G or C preferably. The usual problem is that the 3' end of the primer falls off of the template (this is also what happens during extension at normal extension temperatures).
What's your organism?

Hi phage434, thanks for your good idea. I will try lower annealing temperature. I work on Plasmodium falciparum.
six

Hi, rshi and everyone,
I recently did 5' and 3' race by using RLM RACE kit from Ambion. The goal is to search 5' and 3' end of splicing variants of a gene. The kit seems working but i fail to get the gene I am looking for.

First , I did 5'race with nested PCR with several pairs of gene specific primers. I got one band with a short inner primer (17mers). After cloning and sequence with 5 clones, it is not a gene I am looking for. But I indid find 5' inner primer in this sequence. So at least this kit is working!
The PRC from the rest inner primer only show smears.

The 5' end of full lenght of this gene has ~150 bp GC rich region, so I used PHUSION-DNA polymerase( FINNZYMES) with DMSO with 55oC annealing tamperature. I am about to try it again with GC buffer.
I am looking for help and suggestion.

Second, I also did 3' race with this kit. Two out three species of PCR bands are the splicing gene I am looking for. However, I am not sure whether they are native 3'end the reasons as follews:
1) none of them have 12 polyA as kit indicated. One has 7 polyA, another has 4 of A followed by GAC and another three As.
2) The ending is not at predicted exon dornor side.
Can I trust this result. If not, what else I should do? I am really need the expertise opnion, which Ambion tech support can not anwser at all.

las-sammy