I just found that the user guides of Stepone machines do not recommend the use of SYBR green for the qPCR absence/presence experiment, but I think that my melt curve analysis is good enough to rule out nonspecific amplification. Can anyone tell me whether I am wrong or have missed something important? SYBR green is more convenient for me so that I don't have to order a new probe every time I use a new primer set.
SYBR green is non-specific. So it'll be incorporated into any amplified product. In theory this is OK. It is regularly used for sequencing library quantification.
If you are only looking at presence/absence for one target you might as well design a probe to work alongside. If you are looking at multiple targets then you would have to design new primers for each and therefore its just as easy to design a probe at the same time.
The data you generate will be more robust and sound. Have a look at the Life Sciences Probe qPCR assay search tool. this will give you a good idea on what is best suited for your work.
As far as I understand it, by doing a dissociation curve you are making sure that there is no non-specific amplification. So for a presence/absence experiment surely that is sufficient. If the melt curve shows only one peak (one product) then there is no non-specific amplification. SYBR green is a lot cheaper and simpler so we use it quite a bit. Especially for big GeNorm experiments where we are using multiple genes.
no problem to use SYBR detection unless you want to detect an internal control as well. For non specific product detection indeed you should perform dissociation curve. You can also use a SYBR premix ensuring greater specific amplification like Takara's SYBR Premix ExTaq II (p/n RR820A)
I think that you should include appropriate positive and negative controls in every PCR run. So with a melting curve analysis you will be able to discriminate specific/non specific amplicons.
It is possible to perform presence/absence (and even quantification) experiments with SYBR green. The important thing is to have positive and negative controls and perform and check the melting curve experiments. As soon as your primers were highly specific you will have no problems.
SYBR Green is a non-specific dye, therefore is very important the use of very specific primers, and negative and positive controls when you use it as a DNA binding dye. As well as a good PCR protocol (the higher possible annealing temperature) and a good melting curve analysis. I've'been used SYBR Green to detect the presence of Legionella spp. in different water samples and I've never been greater problems. Maybe you need a more carefully result analysis (ans some experience) than when specific probes are used.
I understand you when you say it is more convenient to you. As far as my experience, yes of course you can use SYBR Green for detection. As the above fellow colleagues mentioned, just make sure you have specific primers, do some blast to ensure their specificity and you definitely need to include controls and if you are so concerned about the results, in addition to the negative and positive control, include Negative Target DNA Control, as well.
No matter you have a good amplification reaction and unique PCR product, this is valid for positive samples. You'll never know if your "negative/absent" sample is a true negative. A NC added to the run doesn't tell you about the inhibition for example.
If you decide using SYBR approach, yes, you run NC and PC controls to make sure that your amplification took place in general, but to release negatives you have to recheck all initially non-detectable samples by a couple of dilutions (1/40, 1/100): if the sample remains negative, it's a true negative; if the sample gets positive in dilution, well, it was inhibited.
Triplicate PCR reaction is worth whether your pipetting precision is questionable. Practicing (and with bit of talent, I think :)) you can reach less intra-assay variability and so be able to run 1 sample-1 reaction. Normally you should fall below 15% deviation either result from the mean (of the triplicate) to allow yourself single reactions. But it's perfectly feasible.
Triplicate also make sense if biological sample is concerned. For example, to have 3 separate extractions from the same sample, this is a meaningful triplicate. Pipetting 3 times the same reaction is nothing more than a technical replicate: yes, Alan has excellent pipetting skills (or not).
It's entirely up to you
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