Fluorophores World : Part 2

Now that you have seen a typical qPCR system using a TaqMan probe, let is go to the next step : Duplex.

First of all : why is it better to do a duplex PCR, that is to say having 2 PCR system in the same well with 2 TaqMan probes?

I could answer you briefly without any clear explanation talking about identification, inhibition, control and genotyping. But you will be surely a bit lost and you will not know more about why and how...

One of the main interest for duplex qPCR is : inhibition control

Indeed, for a trivial reason, PCR is an enzymatic reaction prone to inhibition from different compounds. Inhibition has also a major role in the result given. When a positive result is obtained, there is not a lot of question about it, it is taken like that.

On the other hand, when result is negative, does it come from that there is definitely not any target in the sample (true negative) or because the sample is inhibited and PCR does not work well (false negative). How to know?

Using inhibition control is the answer.
This one is composed by a primer and probe system but also by a target DNA. In order to distinguish both in the same well, a difference has to be exploited. The basis fot that is to use a grafted probe with a really different fluorophore from the target one.

Following combination is obtained so :

target DNA
target primers
target probe grafted by FAM for example.

control DNA
control specific primers
control specific probe grafted  with a different probe than the target one

Among available fluorophores, the range is wide and can be seen for example on provider website like   Biosearch or Eurogentec.

The most used ones are FAM (again) and VIC even if it is not the best combination for optimum use, but it is linked to PCR history when these two molecules were the almost only ones on the market, doing good job for duplex. Today, many other exist...

Let is say that we will use FAM and VIC  fluorophores for our duplex example.
It is very important to check fluorophores overlapping for excitation and emission spectra, because it can have a very big impact on detection signal if not well managed. Obviously, FAM and VIC fluorophores have overlapping spectra that has to be compensated then.


These closed spectra induce interferences between the two signals as we would like to discriminate them.
FAM signal (here in green, and one of the strongest emiting fluorophores on the market) overlap the VIC one (in yellow) in a pretty important part. Compared to TAMRA (a lighter overlapping) and Cy5 (no overlap at all), VIC is strongly interfered by FAM. Both signals will be biased and will give bad results for quantification.

To get rid of this problem, a color compensation has to be made. It is a little amplification to do one for all to help the qPCR device to discriminate between both signals. Normally, device supplier can help you if the program is not mentioned in the device notice.
A compensation run comprises a complete mix with two fluorophores and target nucleic acid, but also all the fluorophores in a single detection. When run is done, it will generate a program helping the software device to better discriminate the signals for any detection then.

VIC signal is pretty often the Inhibition Control (IC) signal. And its signal has to be always the same for the Ct. If IC Ct for any sample is the same as the control one, the sample is not considered as inhibited. If the sample Ct IC undergo a Ct shift of at least 3 Ct (1 log), it means that sample is inhibited and that the quantification or detection made is wrong.
If a partial inhibition, IC signal will be shifted and curves flattened, but still positive.
If a complete inhibition, IC signal is not present anymore.

Here a synthesis of results that can be obtained :

Target	Control	PCR résult	Comments
+	+	Positive that can be quantified	True Positive
+	-	Positive with partial inhibition	Partial Inhibition
-	+	Negative	True negative
-	-	Inhibited	Total Inhibition

In the same well, a detection and quantification can be made for a sample but also insuring the results because of inhibition control avoiding true negative.

As told before, many qPCR duplex are made using FAM-VIC, but need color compensation, whereas many other conbination exists working at least the same with less proble to set up (like Cy5-VIC)

In recent years, Quenchers have been improved to replace useful but fluorescent TAMRA one. New quenchers are non-fluorescent and can also help hybridization of target probe like MGB solution from Life Technologies/Applied Biosystem. Multiplex are easier to set up because no backgournd signal coming from emiting quencher like TAMRA.

Most used ones are :

- BHQ (Black Hole Quenchers) I, II et III , depending on the reporter used
- DDQ (Eclipse Dark Quenchers) : similar to BHQ
- MGB (Minor Groove Binder) : molecule improving probe affinity to DNA and comprising also a NFQ (non fluorescent quencher) developed by ABI.

It has to be noticed that using a duplex need quite often working on qPCR parameters to optimize it like IC DNA to use, primers and probe concentrations, hybridization T°, MgCl2 concentration or adjuvant adding.

More details in another post, but also few words on triplex, fluorophores and tips from my PCR experience.

Have good amplifications....

Fluorophores world : part 1

To understand many things in the qPCR world and to do the right strategic choice, it is better to know few little things about the fantastic world of...fluorophores.

As previously said, PCR systems need primers and if used a probe. I will take a double hybridized probe TaqMan type as an example for this topic. 
These probes are grafted on both extremities by a reporter and a quencher (explained in a previous post)

If using only one probe in the PCR master mix (a simplex), most used fluorophore is then FAM (6-carboyfluorescien or 6-FAM) and the most used quencher is TAMRA (6-carboxy-tetramethyl-rhodamine)

FAM is also one of the most intense molecule considering fluorescence answer to excitation and TAMRA is just the most used for a while but having the drawback to have its own residual fluorescence, able to cause interferences to PCR reaction with small target quantities. TAMRA is replaced more and more by non fluorescent quencher like BHQ (Black Hole Quencher), DDQ (Dark Quencher) or MGB from Applied Biosystem-Life Technologies (comprising a Non fluorescent Quencher).

Hereunder, normalised emission spectra from few fluorophores used in qPCR

It can be observed that FAM and TAMRA spectra are well dissociated and allow a good signal distinction.


In the following table, many more fluorophores are described with their emission and excitation wavelength. Quenchers are also reported on the table side with their use range.

If the table is not clearly visible, here are few links to find any answer about fluorophores association

- IDT report very interesting : http://www.idtdna.com/pages/docs/technical-reports/fluorescence-and-fluorescence-applications.pdf
- Gateway table : http://www.sciencegateway.org/resources/fae1.htm
- Eurogentec guide : http://www.eurogentec.com/uploads/qPCR-guide.pdf

These references are good ones, especially Eurogentec guide. To be honest, I avoid in this blog to repeat one more time things that are described already elsewhere, but sometimes it has to be made. 


We can setup probes having FAM-BHQ1 but not FAM-BHQ3 considering the aborption spectra from the quencher. We can also setup a Cy5-BHQ3 probes but not with BHQ1

Fluorophores choice is essential for a good PCR. It is much more essential when PCR strategy needs a duplex or duplex amplification because chosen ones have to be efficient enough but without having a big wavelength overlapping or too much different fluorescence intensity. 

All of this will be part of part 2.
Have a good PCR.

Thanks and let's carry on...

Hi all qPCR users (or not),

Just few words to thank all visitors to come and read me because it encourages me to carry on.
To be honest, I do a french version for few years and I decided to translate it for non-french speaking people.
Actually, I have started it because when I wanted to find informations about qPCR, I found few tutorials, forums of course, but almost nothing for troubleshooting, special cases or tips to know about the technique.

And even if you are not thousands of people reading my work, you can be up to hundreds per month to come on my blog.
So I will try to carry on translation and update as fast as possible, but do not hesitate as well to send me questions or comments to make this blog even more interactive.

I write actually about inhibition control and how to handle it with commercial kits but also with personal construction for competitive control to mimic as close as possible target system.

Thanks to everyone and see you soon.

Probe strategy : DIY

When the work is done with primers and SybrGreen meaning that PCR answer is good comprising a nice specificity validated with melting curves (single peaks, no shoulders, no close primer-dimer from targeted melting temperatures..), next step is to add a probe to the PCR system and to improve one more time your PCR system.

But talking about probes lead also to talk about different strategies :

First, probes has to be grafted with fluorophores. These molecules has to be wisely chosen depending on your final strategy : simplex (only one target per well) or multiplex (from 2 to 4 different fluorophores in the same well). 4-pex is pretty rare for quantification, we can find some more in genotyping detection.

For example, Eurogentec and Molecular Probes websites provide good table to compare fluorophores spectra you can use for qPCR probes.


In most of the possible strategies about qPCR, there are 2 different molecules called Reporter (fluorescent molecule) and Quencher (molecule that can absorb light energy emitted by Reporter when close enough and  transform it into heat). That is by the way, the FRET principle...
 
When Reporter is excited by a light source, it emits a light but is also immediately quenched and emitted through heat. When the probe is degraded through PCR process, the Reporter is released from the probe, get far from the Quencher and can at least be seen by detection system. 
 
This leads to the following next technology :

  TaqMan(R) probe : single oligonucleotide grafted in 5' by the Reporter and in 3' by the Quencher.
This technology relies on Taq polymerase specificity : an exonucleasic activity during complementary strand transcription. Thus, when the probe hybridize on target sequence, there is still no fluorecence detected because Quencher molecule is still close to Reporter. Because reporter is released in the solution due to Taq exonucleasic actitivity and get far from quencher, fluorescence can then be detected.
This method is the most used in qPCR. 




  "FRET" probes : used mainly by Roche. Actually, this comprises 2 oligonucleotides hybridizing close together. Both are is grafted with one molecule, one is called "donor" and the other one "acceptor". (cf hereunder). Probes place is crucial for a good fluorescence answer. In this method, the two probes are essential to get a signal. When they are well hybridized, they are close enough to operate an energy transfer between them. The donor get the energy and transfer it to acceptor one. Fluorescence will then be detected from this step.



  Molecular Beacon (MB) : 
Another probe type developed by Kramer team in New-York in 1999  (http://www.molecular-beacons.org/). And because their website is very nice, I will not try to explain MB better than them.

MB design is once again crucial for this specific probe strategie but still using the FRET system for energy transfer. As can be seen on next figure, the probe is grafted with a Reporter (green) and a quencher (black). Its very special hairpin structure needs a very specific design in order to get a specific hybridization without interfering with target hybridization. 
When target sequence hybridizes the probe, this one unfold completely to the sequence. In the meantime, Reporter gets further from the Quencher and fluorescence can occur and be detected. This strategy does not imply hydrolysis of the probe by the Taq and can be used again during nect amplification step. 

This strategy work very well and is used especially for genotyping. Fluorescence has to be detected during hybridization step and not elongation if you use 3-step temperature for PCR reaction. 



Scorpions :
This strategy is more primers modification rather than probe strategy. Somewhere, it is a mix between a MB probe grafted on a primer (see figuer hereunder). Very few references about the use of scorpions primers even if part of many oligo supplier catalog.



Few other strategies exist for qPCR application like Uniprimers, but they are not so used in routine application. 
In a next post, I will talk about fluorophore strategies that can be used on probes. 
Have good amplification and see you next time...

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...

Primers and probe design

Here we are…

To be clear, before talking about optimization steps, tests, calibration curves, trueness, validation and inhibition, PCR needs a primer and probe system to work and ensuring targeted gene detection.

So, detection strategy is crucial.

Working in microbiology field with bacteria, viruses and others, I will obviously pick up examples from personal results collected for several years now.

Nevertheless, working on pretty confidential fields in a private company, I will rarely give precised details or goals of the results… I think you will understand…

Let is back to development strategy. Many ways are possible but I can see mainly 2 considering detection and quantification in microbiology field.

The first targets a genus for example, so it has to detect any forms, serotypes or genogroupes and gives a so called “consensus system” even excluding any other microorganism. The second one targets a precised microorganism excluding all the other, one of the most known is Legionella pneumophila.

Thus, considering the 2 strategies, a gene has to be found comprising conserved regions among the different targeted microorganisms (consensus, strategy 1) or a gene comprising mutations in order to discriminate targeted one from all the others.

It foresees many hours looking for good sequences in online database, to compare them and to chose the right one with the right software…or doing it manually with the alignment and operator feeling and knowledge.

Firstable, the NCBI website is inevitable for search of sequences and references.

The approach is simple but can also takes time if meticulous.

1) Search on targeted microorganism in PubMed and also PCR development or genome studies.

2) Find all complete or partial sequences of our target (consensus or specific one).
For example, if you look for Legionella pneumophila on NCBI genome, 45 complete sequences (CDS) will be found. All of them are not Legionella pneumophila because we can have similitude or partial identical sequence. Nevertheless, we can also found the complete Lp1 Philadelphia sequence for example.

3) Back to « Nucleotides » results (more than 3000 items), partial sequences can be also looked for.

4) Once good sequences are found, alignments has to be done to compare them. Many websites are dedicated to it or also softwares like BioEdit
- Multalin
- Clustal software
- A pretty good list is done on Wiki links but as I do not know all of them, be careful about it ("List of sequence alignment software")

5) When a sequence is interesting about alignments and locus, primers and probe has to be determined (next post). Basically, either you have a very powerful up-to-date software like PrimerExpress from ABI, Oligosys or Beacon Designer or you can use free online software like Primer3.

Parameters are complete to change, but you need to know absolutely what you do before changing any of them, because it can affect deeply the obtained results.

6) When primers are chosen, it is good to check with which sequences it can match. Indeed, goals of these oligos is to specifically amplify the target and not the others.

Thus, the use of BLAST (Basic Local Alignment and Search Tool) software permits to check matching one more time of our chosen primers to any other sequences available in the database. It is a good step to check whether sequences are good or not.

Go on "Nucleotide Blast" and then :
- Paste your sequences in the first window

- Name you search in « Job title »
- Indicate the database you want to match your sequence with (on my point of view, it was mainly versus "nucleotide collection nr/nt").
- Optimize your search (in general I work with Blastn)

The obtained results comprises the first 100 results

- Access number of the answer

- Description (name of the matched target)

- Blast scores

- Recovery percentage

- E-value (the smaller, the better..)

If your target is the only 100% found sequence, it is very good.

If not, have a look to the sequences and evaluate the amplification risk of a non-targeted microorganism

7) Another interesting step is to study the chosen sequences with FASTA comparison software.

It will align both sequences and being able to find mutations.

Then, this in silico evaluation is done, and time has come for primers and probes order. Considering probe strategy, it will be discussed in a next post.

Oligos company are present on the market. As for me, I have good results with MWG-Eurofins and Eurogentec.
I always start development with a 0.2µmole order for primers and probes. If you order only primers, you will get them in less than one week.

Next posts will be on primers testing.

Have a good amplification