Primer design - (Oct/10/2013 )
I am new in molecular biology and do not have enough educational background. I try to learn and understand PCR steps. I have questions about primer design.( And I study for a lab where diagnosis of plant viruses were done)
When I look over the sci. papers, the primers were designed to target the coat protein conserved regions of the viruses. As far as I know, conserved sequences are similar sequences that occur within nucleic acid sequences across species or within different molecules produced by the same organism. As I understand a conserved regions can locate in two closely related viruses. For instance, Potato virus A, Potato virus Y.
When I use NCBI conserved region domain for Y and A
GenBANK says coat protein gene is conserved region both PVY and PVA. So I should design my primers in coat protein gene region?
If I do, could it be logical, because they both potyvirus and can share same nucleotide.
But the papers I looked always use primers that were designed to target the coat protein gene of viruses.
I am confused. Can anyone help?
Are you using these primers to confirm the presence or absence of your coat protein? For your virus, does it matter if they primers can differentiate between PVY and PVA?
I use there primers for detection not for differentation. It dosent matter for my virus. I just want to know why primers are designed in coat protein although coat proteins are conserved region for all members of a group (example, potyvirus). I mean, if ı design a primer using coat protein region of PVY, isn't it highly possible to amplify PVA too?
it is possible, but are you looking at a collection of virus' or will your sample contain either PVY and PVA? If your sample contains only one of the virus', it will not be a problem.
I couln't understand why is not a problem. If designed primer for PVY is used for a sample (it can has PVY or PVA or both) gives positive how can I know it is PVY or PVA
If you are studying the virus in a lab setting, chances are that only one variation of the virus (PVY or PVA) is present. If only one is present, you only need one primer as any amplification you see would be from the coat protein of virus which is present.
Even so, are the PVY and PVA coat proteins 100% conserved?