Episode 1: The Importance of Fluorophores & Quenchers in Multiplex PCR Design

Hello and welcome to DNA Software’s first podcast episode with our founder and CEO, Dr. John SantaLucia. Today, we’ll be talking about the importance of fluorophores and quenchers in design in multiplex design and how to appropriately accommodate and account for these different dyes and quenchers. Some of the things I’ve heard from customers over the years, and some of the research that’s been done at DNA Software to consider these. So the first fluorophore question we want to talk about, John is MGBs. I get asked about minor groove binders quite often. And what would be your just general recommendations for people when they design with MGB? What to consider?

Dr. John SantaLucia:

MGBs are interesting. They were developed by Epoch Biosciences way back in about 10, 15 years ago now. They bind to the three prime end of the strand that it’s attached to, and they can move to different locations. So the MGB recognizes a five base pair of stretch, but with the linkers a little bit flexible. So there’s a range of which that MGB can bind to and detect, but the MGBs provide a significant thermodynamic bonus for a perfectly based paired helix. And most importantly, they also provide a discrimination if you have a mismatch, but you need to locate that mismatch near the three prime end of the sequence. So basically that structural distortion caused by that mismatch makes that minor groove binder, bind much less tightly.

So, in cases where you have either a very AT rich target, or if you want to discriminate mismatches for match, the MGBs can be great. Only other limitation is the MGBs work best in a region that has a high content of AT base pairs. So if there’s GC base pairs near the mismatch, then they don’t work as well. But one of the nice things is DNA Software has made our simulation software, so we actually made experimental measurements with MGB with different mismatches in different context and that effect is fully accounted for in our design algorithms.

So just a couple clarifying questions. When you say that there’s a significant thermodynamic bonus, what do we consider significant?

Greater than 10 degree melting temperature enhancement. So a temp typically, 12 to 15 degrees would be very typical melting temperature enhancement for the match. For the mismatch, it decreases the TM by 10 degrees or more, depending on the type of mismatch. So some of the pyrimidine-pyrimidine mismatches are a weaker effect. Some of the purine-purine mismatches are more disturbed, distorting, cause bigger effect. But you don’t need to know that as a user.

The software fully simulates all of that and designs around those problems, to give you the optimal design that discriminates the most.

You anticipated my next question, which is the user doesn’t need to know those TM changes or the thermodynamic improvement from the MGB. But you said something about mismatch and needing to know where that mismatch is. Does the software determined the mismatch or would the user have to specify?

No, you need to specify the location of the SNP site.

Okay.

And then the software would do the design to detect the optimal location of that probe.

Okay. That’s great to know about MGBs. I get asked that pretty frequently. Another question that comes up a lot, certainly when we do multiplex panels is, which dyes to put on the different TaqMan detection probes that are there? Is there any recommendations you’d make? Or I guess having measured this at DNA Software, what are your impressions about the importance of those different dyes and terminal modifications?

Okay. There are several levels of that question. So the first one is the instrument that you’re using yourself is the most important determinant of which fluorophores. So if you’re working with a qPCR instrument that only has one channel, you can only use one fluorophore. And in that case, what would govern your decision would be mainly the brightness of the fluorophore. If you’re trying to do multiplex PCR, so for example, we have an instrument at our company here, the Bio-Rad CFX96, it will detect up to five different dyes. Each of those dyes their wavelengths are separated by about 30 nanometers or more. And so they’re able to achieve a five channel detection within a single well. So really, the hardware that you’re using dictates which fluorophore that you can use for each channel.

It does matter though, the fluorophore choice affects the brightness greatly. So for example, FAM is really bright, other fluorophores can be much weaker like the VIC or HEX dyes are typically significantly weaker. Some of the other ones, Texas Red, can be quite bright and you can see that really well, Quasar 705 is a weaker intensity. So that’s a common issue that users will see, that some channels seem to work better than others, that is a limitation of the quantum yield of the fluorophores, and it’s also a limitation of the instrumentation. The other effects that need to be taken into account, I’ve mentioned there’s multiple aspects to that question, is the thermodynamic effect. So the different dyes contribute differently to the stability of hybridization. So when you’re doing a TaqMan probe, the five-prime end is labeled their fluorophore that can actually stack on top of the helix.

So when the probe lays down the five prime end fluorophore can sort of cap at the end of the helix and form an extra interaction. There’s a stabilizing interaction there due to that fluorophore. Same thing at the three prime end where your quenchers are, they can have a stabilizing effect at the three prime end, for the same reason. They can either stack on top of the helix or sometimes go into one of the grooves to give a stabilizing effect and sometimes a destabilizing effect. So we had NIH funding to measure the effects of fluorophores and quenchers. I think we measured the thermodynamic contributions of over 30 fluorophores and seven different quenchers that are all the most widely used quenchers. So, for quenchers, we did BHQ-1, BHQ-2, BHQ-3, the QSY quenchers, Dabcyl, the Iowa Black quenchers, we did all of those. For the fluorophores we have a very wide range of Alexa dyes, CAL Fluor dyes, CY dyes, FAM, et cetera.

Texas Red, yeah.

Alexa dyes yes, there’s a lot. So we made thermodynamic measurements in many different contexts. We also for molecular beacon design made measurements on a hairpin loop, where you have a fluorophore and a quencher right in close proximity in the hairpin. So we made those measurements with many combinations of fluorophores and the appropriate quenchers that go with those fluorophores. So in that case that a beacon, the fluorophore and the quencher themselves can form a very stabilizing interaction. Actually it’s worth more than a base pair often, but it depends which combination. So again, DNA Software has done all this work for you and it’s incorporated into our software, into Visual OMP, PanelPlex and our other software packages that count for these fluorophore quencher effects.

Yeah. So a reoccurring theme that I hear amongst customers is the sort of thermodynamic knowledge is it might be limited around fluorophores and quenchers, they don’t necessarily know which ones best behave. And the idea is the software will indicate which one to use rather than expecting the user to make a judgment.

Generally, the fluorophore effect could be stabilizing in a few cases, destabilizing, it doesn’t usually affect the outcome of the design in the sense that if the fluorophore is a very stabilizing fluorophore, then the design that comes out will be one base pair shorter. If the fluorophore is a little bit destabilizing, the design might come out one base pair longer th

an you would otherwise have, if you had no fluorophore there.

So in the end, the choice of a fluorophore is not limiting in the design. It’s just something that needs to be accounted for and at DNA Software that’s something we pride ourselves in, making those detailed measurements that allow us to get that supreme super high accuracy of the design. And that becomes important, if you’re trying to do a TaqMan probe, with wild type versus a mutant, and you need to discriminate just a single base pair change, you’re putting the probe on a tipping point. If it’s a wild type, you’re going to get to bind just barely, it’s going to work. If it’s a mutant, then boom, it’s just barely a few degrees melting temperature decrease. And you need that account for those fluorophore effects and quencher effects to get that level of precision to nail it.

So thermodynamic fulcrum, right?

It’s a thermodynamic fulcrum, that’s exactly where we’re at. Where you want to get that probe to be designed very precisely so that you just find, it was like Goldilocks, not too hot, not too cold. You want that TM to be just right where you can detect the wild type and not detect a mutant or vice versa, however you set it up.

Yeah. Okay. Well, these are questions that I hear often. So often in fact, that we thought it was worth sharing in our first episode of our podcast. We appreciate you joining today. And if you have any other questions, you can always email me directly your questions about assay design or multiplex assay design at Joe, joe@dnasoftware.com. Thank you.

Bye everyone.