Help! Help! Need the best experts here!

[bping]

Hi there PCR friends,


I need help designing pcr primers! I HAVE NEVER designed any Pcr primers before.This is part of my MSc project

This is what I want to do:


Part A

1) Design HRM Primers for 2 virus (HSV 1/2)

2)The primers have to prime for both virus species.

3) I will differentiate between HSV1 and HSV2 by melting analysis.

4) I would like to include an internal control that would be primed by the same primers and this DNA can be synthesised.But it has to have a lower melt than HSV 1/2.

5) Include a human housekeeping gene (e.g. actb) to make sure that we have human cells in our sample. I would use HRM too here.


PART B

1) All of the above but using different primers for HSV1 and HSV2.




Where should I start?


I have been on the NCBI database and retrieved the viral sequences.Where to go now?

I would like to learn how to do it and just need pointers in the need direction!

Thanks

Bruno

[danfive]

Find a gene that isn't 100% homologous in both viruses, locate homologous regions within the sequence and design potential primers (you can cut and paste the homologous sequence into online free software, like primerquest, and it will do it for you); pick best candidate primer pairs and try them out in PCR assays.

online tool mentioned --> http://www.idtdna.com/Scitools/Appli...s/Primerquest/

Oh yeah your areas for designing primers should be homologous but the region in between primers should have some base/sequence differences between the two viruses. Go for more than just a few bases differences if possible, actual viruses (not just the sequenced ones) will have occasional mutations.

[bping]

HI there and thanks for the quick reply.

I think I am ok with designing a primer now.My main problem is to pick the gene regions.You said that they shouldnt be 100% homologous.I understand the principle but how can I check these gene regions?

The other problem that I have been thinking about is how can I pick a region of the virus that remains constant throughout the different strains in the same species? Because virus mutate but there are regions that remain costant...

[danfive]

Originally Posted by bping

I understand the principle but how can I check these gene regions?

The other problem that I have been thinking about is how can I pick a region of the virus that remains constant throughout the different strains in the same species?

1st Question. compare them side by side, there's probably special software, but you can use a word processor or text editor. Pick something like a 10-20 base sequence and Use the find tool to find "it" in the other virus' seq. Hi-light and align them, then visually scan to find differences.

2nd Question. Check the literature for gene families and gene homology for that virus family. Once you know the genes you can do other homology comparisons and find the most conserved sequences for the gene-of-interest.
Depending on the family the capsid/protein coat is most likely to tolerate mutations while the replicase(if any)/nucleoprotein genes will be more stable.