nested PCR and quantification by T-RFLP

[valou]

Hi all,

I currently work on arbuscular mycorrizal fungi (AMF) that inhabit plant roots. Because I have very few roots to extract DNA from, I obtain a low DNA concentration after the extraction (beadbeating + DNeasy plant kit from Qiagen). Moreover, my guess is that plant DNA is much more abundant than fungal DNA.

I have tried running PCRs to amplify part of the fungal 18SrDNA (800bp) using primers that are AMF-specific. I used 1, 5 and 10 ul of DNA extracts (more or less corresponding to DNA amounts between 5 and 150 ng) with 0.2mM each dNTP and 0.2uM each primer. No amplification. In another PCR, I doubled the dNTPs and primers concentration. Still nothing.

I am just thinking that the target DNA might be too rare in the DNA extract so I might need to use nested PCR. The issue is that I study AMF communities using T-RFLP. With T-RFLP, the peak area of each T-RF is considered an estimate of the abundance of the T-RF (or OTU). I am wotrying that this assumption might not be valid anymore if I use nested PCR.

What so you think ? Can I still consider T-RFLP as quantitative after nested PCR ?

Thanks !

[Aga]

Well, this makes it all more complicated. Considering the fact that you may have to use nested - PCR for this analysis you will have to know exacly what is the relation of the amount of larger DNA fragment amplified by nested - PCR to the amount resulting by amplification of your 800bp 18S rDNA.

In theory one longer fragment amplified in the first step of nested - PCR should contain one fragment of your target 16S rDNA fragment. [Another problem is when you have several repetitions of target fragment and the number of the repetition varies among different strains, but in case of conserved 16S rDNA region it should not be so.] But PCR efficiency is not 100%.

If you run several repetitions of nested - PCR reaction and establish the relation between the amount of longer sequence and shorter sequence you may use it for your estimation of the abundance of TRFs as long as you have constant relation for the repetitions. You'll need to validate the method though, and it will take some time.

So to make it more clear - after nested - PCR you'll have longer fragment. Next is the second step of nested - PCR resulting in obtaining shorter target sequence (800bp). After T-RFLP you'll obtain a peak with certain fluorescence intensity. Knowing how this intensity corresponds to the amplification in nested - PCR you can probably estimate the abundance of T RF.
However this is my assumption and you'll have to make tests.

[valou]

thanks for your reply !

That's a tricky problem. I could find a few papers where it is stated that it is better not to quantify after nested PCR. So I might have to think of something else :-(