RT-PCR primer design - exon-exon primer design (Oct/15/2004 )
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.
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:
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.
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.
I think you're right. I didn´t considered the exon-intron junction. Thanks for the corrigendum!
I have the same problem and I don't know if it is the fastest and best way I figured out, but I share, it might help you,too.
I search for the gene in Entrez Gene (NCBI homepage) and use the Graph display. With this I can find the exons in the genome and identify where they start. Then I find these sequnces in my FASTA format mRNA sequnce and identify the exon-intron boundaries. It takes quite a lot of time, but since LocusLink has been removed, I haven't found any better way to identify exon-intron boundaries.
If you have any better idea, please share with me.