Help needed with Primer design

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MarkSD135
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Help needed with Primer design

Post by MarkSD135 » Mon Mar 01, 2010 5:49 pm

Hi there,

I'm new here. I'm hoping that finding you guys will get me out of a tight situation :-)

I am having a few problems with preparing for my final Masters Degree project and hope that one of you could possibly point me in the right direction?

Basically, I am looking to create primers for the RAD51 mRNA nucleotide sequence in the yeast Saccharomyces cerevisiae, in order to then monitor the levels of that gene product following various protocols.

The problem is that when I use NCBI, there is no refseq mrna when I find this gene. I can find the DNA nucleotide sequence, but as there is no refseq for this gene I can't make primers specifically for the mRNA, just for the DNA sequence. Obviously with the introns being removed, I run the risk if I try to make primers from the DNA sequence of creating primers for part of the sequence which may be deleted when it is transcribed.

It could be that I am misunderstanding NCBI and am just not using it properly, or that there is just no information on the mRNA sequence for RAD51, however, surely if the site has the protein sequence and gene sequence, then it must be possible to generate the mRNA sequence? I can then order my primers :-)

Any help would be very gratefully accepted.

Take care

Mark

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JackBean
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Post by JackBean » Mon Mar 01, 2010 6:27 pm

http://www.yeastgenome.org/cgi-bin/locu ... ocus=RAD51

next time, try to look for ORF or CDS, which should be also at the DNA sequence in features
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Re: Help needed with Primer design

Post by MarkSD » Tue Mar 02, 2010 7:52 pm

Hi there,

Thanks for the advice. I've tried to work this out myelf, but am still having a couple of issues.

I tried using that site and get the ORF sequence for RAD51, which gives me the below when trying to create primers.

http://www.yeastgenome.org/tmp/sorted.tmp.29829.html

But wouldn't these primers just amplify DNA, not mRNA? Basically I need to create primers for a section of the single stranded mRNA rather than for the DNA.

Any further tips would be very gratefully accepted :-)

Mark

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JackBean
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Post by JackBean » Tue Mar 02, 2010 8:19 pm

for that you must be sure, that you have only RNA (best mRNA), not DNA isolated ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Re: Help needed with Primer design

Post by MarkSD » Wed Mar 03, 2010 1:49 pm

Actually I think I have some primers now.

I was just wondering though, for mRNA primers should they contain Uracil? the primers that I have found don't.

Mark

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JackBean
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Post by JackBean » Wed Mar 03, 2010 3:13 pm

no, you will synthesize cDNA, so no U ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Re: Help needed with Primer design

Post by MarkSD » Wed Mar 03, 2010 4:23 pm

Hi there,

That makes sense. But (and maybe my lack of expertise is showing here) I am still a student after all :-) for the primer to bind to the mRNA effectively, wouldn't it need U's as mRNA contains U's?

Mark

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JackBean
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Post by JackBean » Wed Mar 03, 2010 5:05 pm

mRNA contains Us, but you can have duplexes of two RNAs, two DNAs or DNA and RNA, so don't worry ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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