PCR with more than one primer

[thinkergenetics]

Could someone help me?

Is it possible to perform a pcr with four pair of primers in the same tube, and then, to check in an agarose gel, the products formed?

If possible, wich the parameters that I must to folow?

[r.rosati]

Yes, of course. It's a multiplex PCR.
You should check every primer for dimers against all the others. Or at the very least, the last 3 nucleotides of each primer should not have a complementary match in any of the primers.

[mchlbrmn]

This will not be easy. A problem is that the first PCR product produced will start using up the reagents, such as dNTPs, and creating inorganic phosphate to push the reaction the wrong way (I think), and producing large amounts of PCR product to keep the available enzyme busy, so that by the time the lowest concentration target starts to be produced the amplification may be less efficient.
If you are trying to be quantitative there is the usual problem in conventional endpoint PCR that the concentration of the final PCR product at plateau (end of amplification) is not necessarily related to the initial concentration; factors other than the initial concentration influence the plateau level of PCR product.. qPCR is more accurate in this regard because it reads the concentration when product is first detectable, at a threshold point before plateau even begins.
So, you can do quadraplex conventional PCR, but the weakest targets, or less efficient primers' product, may not be detectable, or may be detected at a lower level.

"Parameters". You use normal PCR parameters, but all the primers must work well with the same program, and it probably will work better if the target concentrations are similar. I suppose that this is the case for single copy genomic genes, with no pseudogenes. Of course the product sizes must be different enough to distinguish on a gel (>10 or 15%, I think), unless you will detect them by hybridizing probes to a blot of the gel.

[thinkergenetics]

I was thinking to do that only to do the first test of my primers. It were primers for amplification after bisulfite treatment and I do not have sure that it work. So I first tought to do a conventional PCR to test them. If I see the bands I will perform four different PCRs to test the best temperature.

Do you think that is a bad idea?

[mchlbrmn]

I didn't entirely understand. "If I see the bands I will perform four different PCRs to test the best temperature": If you see the bands haven't you already tested the temperature?
I haven't done bisulfite treatment. I thought the goal there was to do sequencing of the product?
You can test your primers in the qPCR machine, or at least with the qPCR reagents, so that the conditions will be the same as the final experiment. Oh, on rereading you are not using a realtime machine, perhaps.
Yes, it sounds good to test the reactions separately first. When combined they may not compete equally. For duplex PCR, which I do, I've needed to adjust the concentrations of the primer sets to get both products to be generated equally. I use a full 20 ul volume, while usually I reduce the volume in single PCRs, because the plateau level of each peak is lower, I think. It will be more difficult with four sided PCR, and you probably want to try the maximum volume reaction. When designing primers, I might order more than one primer set for each target gene so that you can find one for each gene that performs optimally under the given conditions. I was going to add that then you could substitute an alternate primer set if two sets interact to create dimers, but I guess you wouldn't be able to tell which primers were interacting.
Maybe you could first try the individual PCRs, then combine both in tetraplex and also in various duplex combinations to give you some idea of how they compete with each other, and how you will need to adjust the primer combinations.
You're not trying to be quantitative, are you? The duplex PCR I did was for genotyping, so all I needed was a yes/no answer, and the amounts present were only ++ +- or --.

[thinkergenetics]

Dear mchlbrmn,

Thank you very much for your comments and my apologises for did not provide all the details before. But how can I test my primers in the pPCR instrument? Is it possible to find the better temperature in a qPCR run?

[mchlbrmn]

I didn't actually know you were doing qPCR. A realtime PCR machine is just another PCR machine; samples can be run in it and removed afterwards to run on an agarose gel, if the melt curve generated does not tell you what you want to know. I suppose that if doing qPCR another issue is that it will be difficult to get 4 products with dissociation (melt) curves that are distinguishable from each other... but, maybe you would do qPCR with probes or other method and not SYBR, and would not have melt curves, since you could get no data for 4 targets from SYBR. I have little experience with probes, but the issue of the probes cross reacting would be another reason they would need to be tested together. But, I agree the first step would be to optimize them separately.

[r.rosati]

Hello thinkergenetics,
by your posts it seems that you're just planning on doing the four amplifications together to save time in checking primers? If this is the case, then you'll have to do them separately, I'm sorry. You can only do a multiplex *after* you know that the individual amplifications are specific and clean.

[relaxin]

Multiplex real-time PCR is possible using TaqMan® probe–based assays, in which each assay has a specific probe labeled with a unique fluorescent dye, resulting in different observed colors for each assay. Real-time PCR instruments can discriminate between the different dyes. The signal from each dye is used to separately quantitate the amount of each target. You have to make sure your qPCR machine has 4-color optical LED recording system.

[thinkergenetics]

Thank you everyone, for the great help.

According your suggestions, I am trying to do the PCR with each primer.

Since I get the results, I will post here.

Thank you very much.