Inserting DNA molecules

A little detailed review with primer testing using Sybr Green intercalating dye for non specific amplification. Here are few explanations about it and other similar solution available :

- SybrGreen : assymetric cyanine that inserts itself into the double strand DNA little groove. When inserted, it emits a fluorescence at 525nm wavelength, usable at FAM channel in a PCR platform. SybrGreen is the most used dye in non specific qPCR and have very strong fluorescence intensity. It gives two different types of information : Target quantity with curves correlated to calibration curves and amplicon quality with melting curves. This last type of curve is obtained by doing a additional amplification program to amplification one (pretty easy to setp tup, ask if you do not find one) and give, after data analysis, the amplicon Tm depending of its size and base composition. Every different amplicon will have a different Tm.

Resolight (Roche) or LC green (for HRM application) : HRM (for High Resolution Melting) is a technology using next generation of intercalating dye inserting more efficiently in the DNA groove than SybrGreen. Indeed, whereas SybrGreen insert itself every 5 to 8 bases among DNA groove, HRM dyes (like LC green) insert in contiguous way and leave no gaps in insertion. Thus, single base mutation or little sequence difference are detected more easily with a fully inserted dye. That is why genotyping has made so huge advance since this technology has appeared. A single mutation can be detected because it will modify the Tm. Used wavelength are around 480nm excitation and 520nm emission.


Boxto : molecule from assymetric cyanines family like BEBO, occurs like Sybr Green. I do not know the intercalating density of this molecule but seems to be similar to SybrGreen. What is the interest so?
Its absorption/emission spectra is not close from FAM one (480/530bm) but closer to HEX/VIC one (520/560nm). This is very interesting used with FAM grafted probes because it allows to do a target amplification with FAM and to check primer-dimer formation and amplicon type adding BOXTO to do a melting curve step. And because emission spectra are different enough, this will not give any problem for analysis considering overlapping. And it WORKS !!!
This molecule can be found at TATAA biocenter (http://www.tataa.com/webshop/Dyes/BOXTO/Detailed-product-flyer.html)

Hereunder a little assay made several months ago. One target but 4 differents intercalating dyes or kits comprising a specific dye :
- Boxto (red) : weak signal but it is normal considering that the analysis here is made with FAM channel instead of VIC normal one.
- Resolight (blue) : from Roche master mix
- MESA Green (black) : Sybr Green solution optimized by Eurogentec
- SybrGreen (green) : from Roche master mix

Figure show us that obtained Tm are almost similar but not completely. Resolight gives the finest values and strong fluorescence having a better sensitivity than the others. Boxto gives good information even not analyzed with the right channel. The two other does not give better results than another.
For next experiments, I will use Resolight more often and also Boxto from time to time with the ability to do melting curve with FAM target system amplified.

Waiting for that, have good curves and be bright...

Primer testing

That is the first article where we will talk really about primers and how to chose the best ones considering the obtained results.

Actually, as said previously, no good PCR without good primers, that is the base.
But how testing primers in qPCR without flurorescent probes? Here are the intercalating DNA dyes domains and non specific qPCR...and among them Sybr Green, LC Green, Resolight, Syto9 and few others cyanins.

In order to test primers, we can use intercalating DNA dyes, fluorescent molecules about to hybridize themselves to double strand DNA coming from PCR reaction itself. They insert in the set up DNA groove and give fluorescent only at this moment. Because fluorescence signal is proportionnal to double strand set up DNA quantity, we have amplification curve relating the signal increase compared to cycle evolution.

Herein an example of a primers system research for a specific target

It can be seen the B system in red and A system in black, targets and concentrations are similar in this experiment.
We could conclude that B system has sooner Ct than A system, meaning that the DNA quantity is better. Nevertheless, B system gets a constant increase of fluorescent signal even without PCR exponential amplification. This shape means a trouble in the reaction between the compounds (primers, thermoprofile to be changed, interaction within PCR kit) and for this, the A system should be preferred.

When I had this trouble of residual increasing fluorescent signal, change of PCR kit supplier always resolved the troubel itself, especially for Sybr Green . The main trouble is that is not only because of one supplier material but from interaction of different compounds.

Once amplification curves are set up, one of the best interest in this expériment is Melting curve. After a short added program added following PCR one (95°C denaturation, temperature drop to 55°C in general corresponding to hybridization then increasing of temperature degree by degree taking in the same time more fluorescent signal information than usually. The so-called Tm is the temperature when half of the amplicons quantity formed by PCR reaction are dissociated.

This Tm is directly linked to amplicon composition to be studied (basis composition, length..) and that is how we should do the difference between two of them. The melting curve analysis is usually made with another part of the software to get a graph with fluorescence decrease on y axis and increasing T) on x axis. Then, a derivative analysis is made from these curves, we get a sort of chromatogram (see under with Sybr Green)

Obtained T° peaks corresponds to set up amplicons. When only one target is made, only one peak is formed (like blue curves on the figure). You can also get peaks and shoulders (red curves) corresponding to targets close to the good one but with genetic sequence different in composition and/or length within a same sample. A last example can be seen, the one of primer-dimers, primers that auto-hybridize and making non specific amplicon (smaller, so with a smaller Tm), here colored in dashed black.

Primer-dimers can be avoided first with the help of adapted design software before ordering. Nevertheless, primer-dimers can still occur in tested systems. They can be seen among Negative control, because they have not got any "competitive" reaction and can occur more easily. When a target amplification occur, they are quite often unseen.

These primers-dimers are easily recognizable because they occur in negative control wells, but also because they have a lower Tm than target system. In the previous figure, dimer Tm is about 82°C for a target system around 89°C.
Hereunder, another system than the previous one but with the same DNA samples tested show us that target curves are better and it remains only one primer-dimer among all the tested negative control (more easy to get rid of adding a probe in the system, but that topic will be discussed in a next post).

In conclusion, it is clever to test at least 2 different system for an identified gene target with different Master Mix PCR supplier. Try not to be stuck with only one supplier or reference, composition and Taq processivity can change drastically your results.
In a next post, I will talk about intercalatinng dyes that I tested in my lab. And, believe me, here again, many difference can occur considering one dye to another.
Have a good curve...