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RT-PCR primer design


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5 replies to this topic

#1 Rosane

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Posted 15 October 2004 - 05:43 AM

Hi

Can you help me?

I need of a program to detect in mRNA sequence the exon junctions. Becouse I need to design primers that donīt regonize genomic DNA.

Thanks

#2 sage

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Posted 15 October 2004 - 04:13 PM

To know exon-intron junctions, the best way is to search genome databases which have annotated such information to most of known genes. Although you can use some program to predict exon-intron junctions by giving a genomic sequence, such prediction may not accurate because it uses much less information than genome database annotation does.

To answer your question about RT-PCR primer design, you can read this post:

http://www.protocol-...?showtopic=2706

Edited by sage, 15 October 2004 - 04:14 PM.


#3 aleruiz

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Posted 27 October 2004 - 06:13 PM

Hi, Rosane.

To design your primers for RT-PCR you can do a search of the ORF of your interest gene. You can select the codifing region and design your primers uppon that sequence. I have had very good results doing this.

Regards.

#4 sage

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Posted 27 October 2004 - 06:47 PM

Hi aleruiz,

I could not agree with you. You have to check the genomic structure of your gene to make sure the RT-PCR primers either span at least one intron or bridge exon-exon junction. You can first design primers using the coding sequence, then check if the amplified region contains any introns.

#5 aleruiz

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Posted 28 October 2004 - 10:13 AM

Hi, sage.

I think you're right. I didnīt considered the exon-intron junction. Thanks for the corrigendum!

Regards.

#6 Gu_Margaret

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Posted 05 November 2009 - 05:14 AM

A new website http://www.mybioinfo...arch_symbol.php allows you to search your gene and have the exon intron displayed in a tabular format, with sequence links of course.
Great tool for designing primers that bridge intron.




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