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Maximum PCR product Tm


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6 replies to this topic

#1 Trof

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Posted 26 September 2012 - 05:33 AM

I've got a 3 primer assay (2 forward, common reverse) for distinguishing mouse gene from human. The product lengths are around 330 and 550 bp and have the same GC content.
I was originaly thinking I could convert it to SYBR assay and distinguish product from melting temperatures. That would be handy for genotyping, because it's quicker.
Well seems that both of the products have same Tm. Also Tm calculators gave me same values so it seems I've reached the product max Tm limit.
I expected there would be one, because you can't have increasing Tm with length forever, but I thought it would be further and I could just use SYBR.

I was trying to find more about this limit, but googling doesn't seem to be much useful, only finds primers all over again, do any of you have some info on this topic (like what length is limiting, what affects it, if it can be modified for products above such length,...)?

Strange enough, when I tried to run up a HRM analysis module on this result, to see if there are any differences in the melting curve shape it didn't showed any, even with my third sample being mix of mouse and human DNA, so the should be at least visible heteroduplexes. Of course SYBR in qPCR concentration in not suitable for HRM, but I'm suprised I don't see any differences at all, we're not talking some 1 bp difference here.

Thinking if there is some way how to see in in real-time without designing new set of primers with shorter amplicons.

Maybe I'll give it a try with HRM dye.
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#2 cellthetruth

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Posted 02 October 2012 - 05:23 AM

Why don't you clone your PCR products into pGEM-Teasy and then sequence it? Then BLAST?

#3 Trof

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Posted 02 October 2012 - 09:36 AM

I don't want to sequence them, I know their sequence, I'm looking for a quick way to genotype new samples (wihout running on gel, which I definitelly can as a simpliest solution).

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#4 hobglobin

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Posted 02 October 2012 - 10:01 AM

Well, "Denaturing Gradient Gel Electrophoresis (DGGE)" and TGGE uses different DNA melting temperatures to distinguish different genotypes (sometimes used in population genetics etc), perhaps you can adapt this technique somehow.
Wikipedia about it

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

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#5 Trof

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Posted 02 October 2012 - 10:47 AM

OMG DGGE! Don't know better spent time than making toxic gradient gels ;)
(Just note.. I'm already decided to make new primers compatible with SYBR, but I was wondering more about the fact of maximum Tm and possibility to affect it.)

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 hobglobin

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Posted 02 October 2012 - 11:33 AM

OMG DGGE! Don't know better spent time than making toxic gradient gels Posted Image
(Just note.. I'm already decided to make new primers compatible with SYBR, but I was wondering more about the fact of maximum Tm and possibility to affect it.)

Well polyacrylamid gels are standard and urea and formamid too...not that toxic and used every day in many labs...anyway for your purposes perhaps more TGGE would fit...
And the important point is that they use melting temps to separate e.g. amplicons from PCR that differ only in one base-pair...

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#7 Trof

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Posted 02 October 2012 - 12:13 PM

Well ever heard of high-resolution melting? ;) We genotype SNPs by that, done in 1 hour, quick, reliable. Compared to this, DGGE is the pain in the ass. However running the gel wouldn't be a problem, since the products have quite different lengths, but I'd rather avoid lenghy gel running, if it could be done better way. I want to try HRM, but having two amplicons that would have metling curves distinguishable by SYBR would be even simplier and cheaper.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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