Why do a nested PCR? (HIV virus) - (Aug/10/2006 )
I have been reading articles on HIV, and have been working on them. There is this question that disturbs me. Why does everyone (every paper I read) does a nested PCR for HIV virus. I really wonder why? even in high viral load patients.
Why can't we just do a 1 step PCR and run a gel? Is there any reasons? to increase specificity?
Just Wonderring... I know Denggi does a nested to confirm it's type. what about other viruses?
I would think that nested PCR is used in this case to increase specificity, or perhaps to verify subtype as you had mentioned.
On HIV mostly nested is done to increase specificity (it's a darn varried virus). Also, it's better to have one methodology to work with, no matter the viral loads in your patients.
Actually, it depends on your application, if you just do a diagnostic PCR, you might have a short PCR that is good enough to do it in a single reaction, but for sequencing purposes, you better go nested for specificity.
People ask me why do I do a nested, and I'll answer them for specificity. But went I run a gel on the first PCR product ... there are no bands! No matter how many times I optimized, but when I continued with a 2nd PCR. The bands just appear! Strange...
the problem might be yield-related? After many cycles of PCR, your amplification efficiency drops.
If you think specificity is not the problem, why not do your outer PCR again on your outer PCR (try several amuonts of your first PCR, even perform dilutions...).