PCR primer design

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roniadam
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PCR primer design

Post by roniadam » Thu Apr 03, 2008 2:26 pm

Hi
I want to design a primer for my sequence and i got one primer compatible with one end (5') and the other is compatible with the middle of my sequence not the 3' end. My question is this ok if I want to amplify this sequence. What about the other parth that is located behind the right primer?

second question:
I predicted two genes from genscan and FGENESH. the first one was ok (i.e.found in genbank). the other gene was composed of Poly A and terminal exon only and I was not able to find any annotated gene in the genbank regarding this gene. My question is this gene a true gene? if yes how can i find it? If no why the programs predict it?

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h2so4hurts
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Post by h2so4hurts » Thu Apr 03, 2008 8:04 pm

1. depends what you're using it for. You won't get the whole gene if the 3' primer isn't at the end or outside of the coding sequence

2. It could be a true gene or a pseudo gene. BLAST the terminal exon. It was found because it has coding sequence followed by a polyA track. Most of the predicted genes are just ESTs that were found by reverse transcribing mRNA with oligoDT.
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roniadam
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Post by roniadam » Fri Apr 04, 2008 2:17 am

h2so4hurts

Thank you very much. It is agreat and convencing answer.

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