5 replies to this topic

[Rosane]

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 don´t regonize genomic DNA.

Thanks

[sage]

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-online.org/forums/inde...?showtopic=2706

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

[aleruiz]

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.

[sage]

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.

[aleruiz]

Hi, sage.

I think you're right. I didn´t considered the exon-intron junction. Thanks for the corrigendum!

Regards.

[Gu_Margaret]

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.