same primers, different product in conventional PCR and qPCR - (Aug/14/2012 )

Hello,

I am new here and I don´t know if this question has already been answered but here it goes.

I have run a qPCR with new primers and I got amplification and nice melting curves. IN addition I loaded an agarose gel and the product had the expected size (around 180 bp)

However, I wanted to sequence the product to be sure and when I run the conventional PCR prior to the purification of the DNA and I loaded a gel to see the size of the product I obtain a huuuge product (1000 bp) what has nothing to do with the product I got in the qPCR

Any hint about what can be?? (sorry if this is too basic but I´m quite new...)

Thanks!

Did you also get the 180 bp product on the gel with the conventional PCR?

Were the cycling parameters the same for both PCRs?

It may be that there is some genomic DNA carry over in the PCR that is amplified by the ordinary PCR but not the qPCR (this is usually due to different cycle parameters).

Thx for answering!

No, in the conventional PCR I only got the 1000 bp.

Cycling parameters are indeed not the same (although the annealing temperature it is).

But the thing is that I am amplifying two receptors at the same time with the same samples and this problem only occurs with one...(receptor B works perfectly, I obtain the same product either with the conventional or the qPCR, but A is the problematic one...)

I mean...if there was genomic DNA carryover, shouldn´t I also observe a different product with the other receptor as well?? (Sorry, sorry if it is too obvious but I am very new with this stuff )

Thanks!

Do you know about intron spanning primers? Usually for qPCR you use primers that span intron/exon boundaries, which are usually big enough (500-1000's bp) that with the short extension times in qPCR, they won't produce (detectable!) product - I suspect you have a 1-3 min extension time on your ordinary PCR or are using a proof reading polymerase with high processivity.

Ufff...I didn´t know about that...The extension time in my conventional PCR protocol is 0.5 min but I am using Hot Start Taq Polymerase...(I will check whether it is proof reading, I think it is). But any idea why I don´t get the product of 180 bp?? The only way to resolve the problem is to change the polymerase or is there something else I can do?


lots of thanks! (Im quite lost and my lab mates did never had such kind of problems!)

Hot start Taq is not proof reading, none of the Taq polymerase family are, they merely have a heat labile antibody incorporated with them so that at temperatures below a certain point, the antibody will bind and prevent the Taq from working, hence the "hot start".

Apparently Taq is a bit quicker than I thought; about 50-100 bp/sec added to sequences during extension - so you could easily amplify a 1000-1500 bp fragment with a 30 second extension.

I don't know why you aren't getting the 180 bp product, the only thing I can come up with is that the primers are not ideal for the product you are trying to amplify, but may be for the 1000 bp product.

Well just a quick thought.. since you're not using proof reading polymerase anyway (but I Use HotStarTaq on all sequencing though) , why don't you just put your 180 bp qPCR product on gel and cut it out and sequence?
You want to sequence what you get on real-time, so logical step is to sequence THE real-time product, not some other.

In the meantime you can solve your 1000bp problem. It's very unlikely you would get preferential 1k product and no 180bp one by changed conditions. HotStarTaq is indeed capable to get this 180 bp in 10 second extension, so anything longer is unnecessary. Also you have to take into account that SYBR chemistry inhibits PCR, the mixes can have different Mg amount and so, so the reaction conditions are different, try to optimise Tm for this classic PCR, if something changes.
And one thing I would do, check if I'm really using the right primers