A bit large qPCR amplicon

[Marco A Martinez]

Hi, everyone!

I know this has been asked before and that there's a los of info out there, but I have a very specific question.

I know that the recommended size for a qPCR amplicon lays between 50-200 bp... but I have designed a pair of primers to amplify a 211 bp amplicon... and I don't know, is it too big already?

I really have tried to shot it down, but I just don't get it right, and they don't seem that large to me... do you guys think they could work?

PD: also, I have another pair that amplifies a 203 bp amplicon, and the questin applies the same for them.

Thanks for the help! =D

[namishu95]

it is better to have real time PCR amplicon less than 200bp, but understood, we can't find good primer/probe for some situation, i also have couple sets with amplicon just above 200bp, it works fine. But the problem is the SYBR green result for checking your primer pairs is awful. It is not that easy to check primer dimmer by SYBR green assay. Another problem is the PCR sensitivity with longer amplicon may be lower, it is better to check the assay sensitivity with the other set or technique, I am working on typing/subtyping influenza viruses, we designed longer PCR for H7 subtype, but I did compare to influenza typing assay, the H7 subtyping assay is comparable to infA type assay.

[Marco A Martinez]

What do you mean by an awful checking or primer dimmer? I've read thar I may get a bimodal peak curve, but it didn't seem so bad when I read it... is that the problem you're talking about, or is there something else?

Also, I am working in a diangostic test, so the sensibility is pretty imporant for the assay. Do you it could be improved by extending the polymerization time?

[namishu95]

SYBR Green usually works with amplicon between 100-150 bp, the data that SYBR green assay generated by over 200 bp amplicon is useless.
I don’t think the PCR sensitivity can be improved with extending the polymerization time. it mainly depends on primer-probe design and secondary structure.

[butters]

Marco, if you still read this.

We will never know until you have get the primer pair and test it out. For me, 200++ (just in case if ppl are thinking over 300 or 400 or 500++ bp, no I don't meant that high) is still good to try if really no other option available.

Since you mentioned bimodal peak curve, I would have to think that your rt PCR machine can do melt curve. That would help but it is not definitive. But it is a guide. For example in your "interpretation of result: you need to state the range value of your said product is positive and everything else is negative".

Bimodal or anything more than 2 peak would be attribute to formation of primer dimer. Unless your primer pair is really that BAD, if not you could still do some optimization. Do consult your rt pcr manual for steps on optimization.

Are you making a primer pair with both unique seq or one is unique follow by another not so unique?

[Marco A Martinez]

Originally Posted by butters

Marco, if you still read this.

We will never know until you have get the primer pair and test it out. For me, 200++ (just in case if ppl are thinking over 300 or 400 or 500++ bp, no I don't meant that high) is still good to try if really no other option available.

Since you mentioned bimodal peak curve, I would have to think that your rt PCR machine can do melt curve. That would help but it is not definitive. But it is a guide. For example in your "interpretation of result: you need to state the range value of your said product is positive and everything else is negative".

Bimodal or anything more than 2 peak would be attribute to formation of primer dimer. Unless your primer pair is really that BAD, if not you could still do some optimization. Do consult your rt pcr manual for steps on optimization.

Are you making a primer pair with both unique seq or one is unique follow by another not so unique?

Yeah, I'm still reading, and I will at least until I have them and tell you guys how it all worked out.

I've read in the last week that it's very good to check the PCR product with melting curves and electrophoresis, and I plan to do so. In that way, I could see if the bimodal curve is a result of dimerization or just heterogeneity to melt. And I hope my primers won't be so bad, they have some nice DG values for either hairpin, homodimer and heterodimer... so I really hope that won't be a problem. I will consult the manual, though.

Originally Posted by namishu95

SYBR Green usually works with amplicon between 100-150 bp, the data that SYBR green assay generated by over 200 bp amplicon is useless.
I don’t think the PCR sensitivity can be improved with extending the polymerization time. it mainly depends on primer-probe design and secondary structure.

I'm sorry, I'm a little slow and I want to be sure that I'm understanding. Do you mean the data will be useless as if I shouldn't even try it out?
I'm not really into the quantification, although it would be nice. I kinda only need to know if there was any amplification or if there wasn't... don't you think it could work?



I really appreciate the help of both of you, I just wanna be sure that I'm really getting what you say.

[Marco A Martinez]

Originally Posted by butters

Are you making a primer pair with both unique seq or one is unique follow by another not so unique?

I forgot to answer this. Mmmhhh... I designed generic primers for all pathogenic species of Leptospira... so they're not precisely unique, as all Leptospira spp. contain them... but I BLASTed them to make sure no other thing could amplify with those primers... Anyway, almost every species have at least onw mismatch in either primer... I do understand that will likely reduce even more the sensibility of the assay

[namishu95]

For development of real-time pcr assay, I usually check primers( sensitivity and dimmer) by SYBR green first, if it works fine , I will set Taqman assay to test primer-probe. But the SYBR green results for over 200 bp amplicon were not consist with Taqman assay ( same primers) , for instance I got low Ct value with Taqman assay but pretty high Ct value by using SYBR green .
SYBR green seems to have greater affinity for AT-rich than GC-rich DNA, but ideal amplicon is 100-150bpforSYBR green assay.

[namishu95]

it should be OK to amplify the samples with one mismatch on primers region unless the mismatch is on first 3 nucleotide position from 3' end of primers, but it also depends on what mismatch even it happened on 3' end, such as G-T binding is still pretty stable.
Probe annealing to template with two mismatches was reduced as compared to the matching template and lead to a reduced efficiency and sensitivity of the assay. No signal was detected when the target contained three or more mismatches.