designning primers

[jajo]

Hello everyone,
I am an undergraduate student. I'm doing project on leishmaniasis( chitinase gene expression on leishmania). Before I start the lab, i need to design my primers for RT-PCR. I have already started using the EBI nad NCBI sites. But I have got confusion what I am doing. So, I need some help from people who has experience on this area. Let explained what I have done so far; From the NCBI site, I picked the chitinase gene sequence of leishmania and other 4 different organisms. Then, I changed it into FASTA format, and using Clustalx software, I did multiple alignment,,,and I stucked here. Even I am not 100% sure what I have done is RIGHT. So,PLEASE I NEED SOME HELP.

Thanks a lot

[canalon]

Find areas that are conserved in leishmania not in the other. Depending on how much similarity there is you can either use that to put your primers (use primer3 available on NCBI website) or only the probe.

[JackBean]

question is, whether you need to distinguish between chitinase from leishmania and from other organisms. If so, focus on the areas, which differ, if not, you can take any part of the gene.

[andresgutierrez]

Perhaps this video may help you but the primers design requiere a lot of attention.

http://www.youtube.com/watch?v=6thmaB0liXo

Some advices,

1. Select a conserved regions from your alignment.
2. A good primers sequences has: 18 - 22 nt, 55°C as melting temperature, without second structures but If have it be careful that they occurs under the PCR tempetures. Pay attention if you have degenerate regions.
3. Manual desing is the best strategy for you so I strong recommed to you primer3 and genrunner tools.

But this is only an introduction about your question I can help you If you want so do not hesiate to contact to me.

Regards,

Andres

[jajo]

Thankyou so much u all. It's really helpfull.

[wilsontrace]

Hello,

Multiple sequence alignment won't be required when you need to analyze gene expression of a specific gene. You would need to perform alignment only when you would have had to distinguish between chitinase gene from leishmania and from other organisms.

You may design specific primers directly on the sequences which you extracted from NCBI.
For designing highly specific real time PCR primers for your chitinase gene sequence, try using Beacon Designer from Premier Biosoft - http://www.premierbiosoft.com/molecular ... index.html

Wilson