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Multiplex PCR question

  • 07-07-2019 10:51pm
    #1
    Registered Users Posts: 112 ✭✭


    It might be a long shot to expect an answer to this here. I'm trying to understand Multiplex PCR. I would love if my question could be answered in the context of the video below.

    From watching this video I'm getting the idea that Multiplex PCR isn't quite the the same principle as qPCR. This seemed to be the only clear explanation of Multiplex PCR that I could find, however it is only a particular company's way of doing it so it might be different from the standard Multiplex PCR.

    Anyway, in qPCR, the probe is completely degraded, but in this video we see that the probe is not degraded at all for example. I'm imagining that the strand shown at 0:57 on the bottom is part of the PCR target DNA (after denaturation), and that the part in blue (on the bottom strand), is just the target for the probe. So one question I have is, if the "target specific melting probe" gives out light the very moment it is released from the DNA by the polymerase, then doesn't this mean that each of these probes will give out light at two different times? The second time being when the quenching probe is released, as we see at 1:34 with middle probe on the diagram.

    Am I right in saying that the other two probes on the diagram (far left and far right), are the probes that never got to anneal in the first place?

    https://www.youtube.com/watch?v=DZ7yHQAJBnw

    I'm assuming that in general, with Multiplex PCR, that in order to differentiate between fluorescence from different genes, that the light from probes associated with different genes are of different specific values.


Comments

  • Moderators, Science, Health & Environment Moderators Posts: 4,677 Mod ✭✭✭✭Tree


    Mutliplex just means that you are running more than one primer set in a reaction, you can do a boring gel analysis either to separate a bunch of fragments of different sizes (you would need to know your products). Doing a multiplex qPCR usually means you have a different dye associated with each primer set you are measuring.

    The quencher/dye interaction is based on distance, when you take the quencher away from the dye, the dye can fluoresce. If you put the quencher back, the signal goes away.

    I'm not sure which diagram you are referring to. In the video, there's a dye and quencher on the probe, when the polymerase reaches the primer it cleaves the quencher and leaves your dye free to fluoresce (during the measurement step of the PCR cycle, you don't do it all the time for risk of bleaching it).


  • Moderators, Sports Moderators Posts: 24,413 Mod ✭✭✭✭CramCycle


    Multiplex qPCR and qPCR are identical, as Tree says, you have a bit of optimisation so you can run the same program for both but it just means you have multiple qPCR reaction with different fluorophores in the same tube. It is a great way to save money and get shed loads more data if you design your own. You can get several companies to produce them quite cheap if you provide the sequences.


  • Banned (with Prison Access) Posts: 386 ✭✭Problem Of Motivation


    Tree wrote: »
    Mutliplex just means that you are running more than one primer set in a reaction
    Wow, interesting stuff.


  • Registered Users Posts: 112 ✭✭Game of Thrones Fan


    This whole thing can be quite hard to understand, and I've no doubt it's not all that complicated after all. I'd feel better if you could watch the video, and answer the questions my way! I hope that would be easy for you to do... assuming you're well versed in the area. I don't mean to irritate.

    By now I've completely forgotten why I asked what I did, but when I first typed this thread I made the point to pinpoint each part that didn't make sense to me.

    Below is a definition of qPCR as I understand it, that I have noted in my copy book. This video seemed to contradict my understanding.

    qPCR - This involves the use of oligonucleotude probes that allows progress of the reaction to be monitored as it occurs in real time. The probe binds onto one of the strands on the target DNA. There's a dye on each end of the probe; reporter dye, & a quencher dye. The taq polymerase isn't blocked when the probe & the probe gets digested by it, as the polymerase has 5' exonuclease. When it gets digested, the reporter is freed from the quencher so it can emit light. A greater amount of fluorescence occurs with a greater the amount of cycles. After a certain amount of cycles the reactions will no longer be quantitative due to limiting reaction reagents.
    Tree wrote: »
    I'm not sure which diagram you are referring to.
    Didn't I put time stamps in?
    Tree wrote: »
    In the video, there's a dye and quencher on the probe
    The word quencher isn't even mentioned in this video! It seems as if they're calling it a fluorophore! Also, I haven't heard of a melting domain!
    Tree wrote: »
    Mutliplex just means that you are running more than one primer set in a reaction
    With all due respect, I get that much.


  • Registered Users Posts: 962 ✭✭✭darjeeling


    The key distinction of multiplex PCR is just that you amplify multiple independent targets from within your larger DNA template, through use of multiple sets of PCR primers.

    In any kind of PCR, there are multiple possible uses and consenquently many different ways of getting a read-out. You could determine the size of the product on a gel, obtain the DNA sequence, quantitate using qPCR etc.
    However, when you have multiple targets in the mix, you need to find a way to make sure that you can get a separate read-out from each, so that the signals from multiple targets don't pile up on top of each other in an unintelligible mess.

    E.g. if your assay is to detect subtle differences between individuals in the size of a microsatellite for DNA fingerprinting, you could multiplex multiple microsatellites so long as each has a different size range and there is no overlap between them.

    A quick look at the video you linked shows a multiplex qPCR method for detecting and potentially quantitating abundance of pathogens.
    The assay seems to work by ensuring that each pathogen yields a product with a different melting temperature, meaning that each generates a separate discrete peak in the melting curve read-out.

    Nowadays multiplex PCR is increasingly being used in conjunction with next generation sequencing for high throughput genotyping of tens to hundreds of genetic markers in hundreds or thousands of individuals.
    Multiple genetic loci can be amplified at once, and then millions of individual DNA molecules from the resulting mixture can be isolated and sequenced, allowing you to identify the locus from the sequence and additionally to detect any variation within it that can be used to characterise an individual.

    Problems with multiplexing include cross-hybridisation between primers or their amplicons, resulting in all sorts of artefact sequences that reduce your ability to make sense of the output. Careful primer design and pilot experiments are needed to get round this.


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