Using real-time to differentiate between SNPs - (Jun/03/2008 )

Hi,
Hopefully someone can help me with this one.

We have mutated a virus to carry a mutation and we want to use real-time to quantitatively differentiate between the two populations i.e. w/t and mutated.

I have looked in the literature however somer people seem to use 2 taqman probes, whereas others use SYBR green & melt curves to differentiate between the two different populations - there seems to be no general concensus!

Can anyone either say which is better, or point me to a paper which tackles this.

Many thanks

Quite agree with the previous post. I would go for TaqMan assay. Alternatively, you could try High Resolution Melting software (also ABI).

Really quantitative results you get only with Taqman probes.
Melting curve and HRM could be both unreliable when you got a sample where are both, wt and mutated versions. Melting curve or HRM is good to detect mutations, but not to quantify them from a mix, it's only semi-quantitative at best. But if you don't have "mixed" samples it could work though.

Other thing is specificity, melting curve only distinguish between quite different sequences. HRM can, when optimised, detect even single-base difference.
When you need to quantify one-base mutation with two Taqman probes, it's a need to have LNA spiked probes, and that's even more expensive.

Thanks for the feedback
Think i will go with Taqman for moment - seems more reliable.

I would go for Hybridization Probes - combines the advantage of TaqMan and Melting Curve...
sorry for my late reply.