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PCR primer design

PCR primer design - PCR - Polymerase Chain Reaction Forum

PCR primer design - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 06-19-2009, 12:33 PM
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Default PCR primer design



Does anybody know why we should pick up primers near the 3' end of the CDS ?

Thanks a lot
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Old 06-22-2009, 01:27 AM
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Default Re: PCR primer design

what do you mean by pick up primers near the 3' end of the CDS?
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Old 06-22-2009, 05:11 AM
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Default Re: PCR primer design

I mean the location of the primers in the sequence, I read the primer pair should be near the 3' end. The CDS "coding sequence" of my gene is 1779 bp, the primer pair should be within the range 1200-1500 bp. Do you know why ?

Thanks for your time
Have a great day
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Old 06-22-2009, 06:34 AM
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Default Re: PCR primer design

hmm... I do get what you mean now but I am unsure as to why it primer pair should be near the 3' end. What I do know is that if I want to amplify a region, my primer pairs will be at the ends of this region. The forward being the 5' end of the region and the reverse being the complementary of the 3' end of the region. I am sorry for not helping out much.
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Old 06-22-2009, 02:53 PM
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Default Re: PCR primer design

It depends on what you are doing with your sequences. Are you interested in sequencing the entire CDS of the gene, or only a portion of it? Are you trying to specifically target the 3' end?

Typically, you want to design your primers in an area of the gene that is highly conserved. In other words, if you design a primer in an area that is highly variable, it may not anneal to template- especially if you are studying numerous species. Perhaps with the specific gene you are studying, the 3' is ideal for this reasons.
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Old 06-22-2009, 09:58 PM
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Default Re: PCR primer design

Are you amplifying from a cDNA library? A cDNA library which is constructed using only poly(T) primers is biased towards amplifying only the 3' sides of the RNA library, especially for longer sequences. Use of random hexamers in combination with poly(T) can prevent this. There is to my knowledge no other general rule that states you should amplify only towards the 3' side of genes.

What gene are you looking at and what do you want to do woth it? Could be that this part of the gene simply contains the functional domain of the protein it encodes and as such is the most conserved/interesting for your experiment.

Last edited by Mathijs; 06-22-2009 at 10:04 PM.
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Old 06-23-2009, 12:30 PM
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Default Re: PCR primer design

am amplifying from a cDNA library for RT-PCR. I use random hexamers cDNA.
Thank you all eas really helpful
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