Need to get 3' end sequence! - 3' end sequencing (Jun/18/2007 )
Hello all,
Is there any way to get the 3' end of my mRNA sequenced besides 3'RACE? I need to get full length sequence for my gene and have not been able to get the 3' end yet. I have tried 3' RACE and have not gotten it to work maybe because my expression if too low. Does anyone have any suggestions on how I could get this sequence?
Thanks
I dont think I believe this... The way PCR works these days you would have to be making cDNA from very miniscule amounts of RNA or from an RNA prep where like less than 1% of the cells express your gene for it to not be detectable by PCR. I think you need to optimize your 3' end RACE, possibly try a two-step reaction with nested primers...
these days you can sequence a PCR product, and in general if you can see a band on a gel then you can get sequence from it... if you are still not getting enough PCR product or if it is not pure then clone your amplimer into a vector like pGEM T-easy then you can grow up more than enough for sequencing reactions...
these days you can sequence a PCR product, and in general if you can see a band on a gel then you can get sequence from it... if you are still not getting enough PCR product or if it is not pure then clone your amplimer into a vector like pGEM T-easy then you can grow up more than enough for sequencing reactions...
Thanks for your reply! I'll tell you what I have tried so far and I would love to hear your thoughts. I have isolated total RNA from tissue and used that to perform my RACE reverse transcriptase reaction and I did not get any products when I amplified using my gene specific primer. I also used that same total RNA and primed with random as well oligo dT primers (2 different reactions) when I did the RT reaction and I get products with both of these when I amplify with my GSPs. I have also used the cDNA generated with my RACE kit and amplified it with a housekeeping GSPs and I get product. It is only using the RACE cDNA and my GSP that I do not get any product. As I'm sure you can tell, I am new to this protocol so I would appreciate any help you can give. I am working with baboon and I need to know the full length sequence of my gene as it has not been described as of yet. The human sequence is known and is quite similar to baboon so I also thought about using the 3' UTR human sequence and designing primers to amplify my last exon and get the sequence I need. Any thoughts?
these days you can sequence a PCR product, and in general if you can see a band on a gel then you can get sequence from it... if you are still not getting enough PCR product or if it is not pure then clone your amplimer into a vector like pGEM T-easy then you can grow up more than enough for sequencing reactions...
Thanks for your reply! I'll tell you what I have tried so far and I would love to hear your thoughts. I have isolated total RNA from tissue and used that to perform my RACE reverse transcriptase reaction and I did not get any products when I amplified using my gene specific primer. I also used that same total RNA and primed with random as well oligo dT primers (2 different reactions) when I did the RT reaction and I get products with both of these when I amplify with my GSPs. I have also used the cDNA generated with my RACE kit and amplified it with a housekeeping GSPs and I get product. It is only using the RACE cDNA and my GSP that I do not get any product. As I'm sure you can tell, I am new to this protocol so I would appreciate any help you can give. I am working with baboon and I need to know the full length sequence of my gene as it has not been described as of yet. The human sequence is known and is quite similar to baboon so I also thought about using the 3' UTR human sequence and designing primers to amplify my last exon and get the sequence I need. Any thoughts?
Hi Jennifer, I agree with beccaff in that you probably need to optimize your 3' race. Before I did anything however, I would blast your sequence against the EST database at NCBI (if there is one for baboon?) to see if any evidence is there to suggest that this last exon is transcribed. If so, your golden and you can use that to make GSPs but I would also do a northern to check for other sizes and to assure yourself you have the predominant one. If you've done that already, another possiblity is that you may be getting specific bands but they are smeared, meaning that there are multiple sizes that are amplified. This occurs because the oligo dT adaptor primer will anneal throughout the entire poly(A) tail of your transcript. First I would stain your gel with Sypro to see if you do get some smearing. Make sure the minimal size is equal to the theoretical size predicted from the human sequence. If so, cut it out, purify, clone and sequence. If this doesn't work, I would use LM-PAT (ligase mediated poly(A) tail test) to force the PCR amplification to the very ends of the poly(A) tail which will make your amplicons more apparent. Good luck.
Mateo
Hi Jennifer, I agree with beccaff in that you probably need to optimize your 3' race. Before I did anything however, I would blast your sequence against the EST database at NCBI (if there is one for baboon?) to see if any evidence is there to suggest that this last exon is transcribed. If so, your golden and you can use that to make GSPs but I would also do a northern to check for other sizes and to assure yourself you have the predominant one. If you've done that already, another possiblity is that you may be getting specific bands but they are smeared, meaning that there are multiple sizes that are amplified. This occurs because the oligo dT adaptor primer will anneal throughout the entire poly(A) tail of your transcript. First I would stain your gel with Sypro to see if you do get some smearing. Make sure the minimal size is equal to the theoretical size predicted from the human sequence. If so, cut it out, purify, clone and sequence. If this doesn't work, I would use LM-PAT (ligase mediated poly(A) tail test) to force the PCR amplification to the very ends of the poly(A) tail which will make your amplicons more apparent. Good luck.
Mateo
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Thanks Mateo! I'll give your suggestions a try. Sounds like you have done this before. There is not an EST database for baboon and that is part of my problem. The human and baboon orthologs are highly homologous in most areas but there are enough differences to make this problematic. The last exon in the human gene is mostly untranslated with about 90% comprising the UTR but can't say for sure if that is the case with the baboon. Again, thanks for your suggestions. They are most appreciated.