I want to amplifiy 10 amplicons by multiplex PCR. They should be sequenced on Ion Torrent so they are 180-210 bp long. The amplicons differ for only few bp each other. Standard agarose gel does not have enough resolution. We don't have 2100 Bioanalyzer in the lab. How can check fast and easily if I have all my PCR products? Any ideas?
Do you have a real time thermocycler?
A faster and more precise method than gel electrophoresis is the dissociation curve; with your reaction you have to add SYBR Green, Eva Green or any non-specific dye for DNA; after your PCR reaction, you have to run a cycle that varies temperature from 50-90°C in 3-5 minutes and measure the derivative of fluorescence with respect of time (-dF/dt) versus temperature, you should get results like the figure 16 in the next link:
http://old.padil.gov.au/pbt/index.php?q=node/28&pbtID=168
The advantage of this method is that you can identify amplicons with only one base pair different and you can measure the relative concentration of an amplicon by the calculation of the area below the curve of (-dF/dt) vs temperature graph.
Disadvantages: the technique is so sensitive that you must use a high fidelity polymerase and the micropipeting technique should excel, it´s a big jump between final point and real time PCR in terms of micropipeting technique
Maybe you can design a SNuPE-assay to detect the various amplicons. (You will need a capillary electrophoresis system).
You may use SSCP as an alternative approach.
If you would like to amplify 10 amplicons in one end-point PCR, you will have huge competition for polymerase/dNTP/magnesium ions. Some from those 10 amplicons may not appear. Did you consider to split it into more reactions?
Do you have a real time thermocycler?
A faster and more precise method than gel electrophoresis is the dissociation curve; with your reaction you have to add SYBR Green, Eva Green or any non-specific dye for DNA; after your PCR reaction, you have to run a cycle that varies temperature from 50-90°C in 3-5 minutes and measure the derivative of fluorescence with respect of time (-dF/dt) versus temperature, you should get results like the figure 16 in the next link:
http://old.padil.gov.au/pbt/index.php?q=node/28&pbtID=168
The advantage of this method is that you can identify amplicons with only one base pair different and you can measure the relative concentration of an amplicon by the calculation of the area below the curve of (-dF/dt) vs temperature graph.
Disadvantages: the technique is so sensitive that you must use a high fidelity polymerase and the micropipeting technique should excel, it´s a big jump between final point and real time PCR in terms of micropipeting technique
Either u can use metaphore agarose or PAGE but use the higher percentage of acrylamide may be 12 to 14 %
In my opinion RT-PCR and dissociation curve is not a good solution if you have so many fragments with similar sizes (hence similar dissociation temperatures). I would use denaturing polyacrylamide (6%, 19:1) and silver staining. It's fast and cheap solution to your problem. Take care :-)
But i think you can use the rt pcr dissociation curve also if all the amplicons are differing in gc content
Indeed, the dissociation curve performed in the new generation real-time thermocyclers has the sensitivity to diferentiate between amplicons differing only a base pair long or with a SNP; faster than any electrophoresis technique, more sensitive and by the way @Marcin Wozniak, you forget to mention that the denaturating polyacrylamide should have a gradient of concentration of the denaturing agent to separate the amplicons with a better resolution, disadvantage the extra work time and if you have the RT thermocycler is not an expensive treatment, you only have to buy an extra DNA dye
You can use PAGE for separating the fragments with miner difference in size. If the fragments are similar in size you can use DGGE for differentiating all the fragments. Good Luck..
If you do the dissociation curve I suggest you use a saturating dye, such as EvaGreen. Non-saturatings such as SYBR Green are not as accurate at a SNP level because the fluorescent dye can reincorporate.
I think you should run your amplicons with RFLP marker between 180-210bp and possibly check which enzymes can cut these different fragments
OK, perhaps the new RT-PCR equipement allows for dissociation curve analysis of 1bp difeereneces but as far as I know it strongly depends on the fragment size and additionally may incur some additional cost. For instance, we have ViiA7 machine in our lab but we would have to pay additionally for HRM application. If Paraskevas latropulos has access to such equipement, it's worth trying maybe. However, PAGE is still quite a simple method , the equipement is present in virtually all las and it does not necessarily requires gradient for good separation. I would test denaturing PAGE the separation with single fargments, then mix them in equimolar ratio and separate again to check for resolution. Then one can use such a mixture as an external standard against experimantal samples. Optimizing multiplex RT-PCR reaction may be cumbersome also...
The RT-PCR, High Resolution Melting Curve analysis is able to discriminate SNP between strands that are of identical amplicon size (ideally 70-150 bp).
I can confirm Marcin's statement about needing specialized equipment. We use the Rotorgene series machines and found that the one in our core facilities did not have the necessary filters installed to run HRMA, and because we didn't have the filters the machine wouldn't run the same sort of difference analysis at a normal resolution. These filters (at least Rotorgene) had to be installed at manufacture and do not come after market. Fortunately, once you have the machine and the reagent analysis becomes relatively cheaper. Premade kits come in around $100 and are non-specific (you can use different primer sets), where as restriction enzymes are about the same cost but are specific to one type of cut site.
On the other hand if your amplicons are significantly different in composition and of different sizes standard melting curve analysis should be able to give you usuable results.
Thank you all for your useful advices and comments!
Today I set up the first multiplex pcr (the single PCR were already ok). Tomorrow I will try with the Melt Curve Analysis. We have the 7300 real time pcr of Applied Biosystems that has the dissociation curve. I don't know if it suitable for HRMA but I will try anyway. I plan to use the SYBR green 2x Mastermix. Unfortunately the pure dyes I have are ethidium and SYBR Gold. I hope it will work. If it will give promising results, I will buy EvaGreen.
The polyacrylamide solution with silver staining looks also nice but the protocol I know (for SSCP) is one day long. Is there a faster protocol?
Thanks again. I will let you know...
best technique to discriminate between such fragments is higher percent PAGE and silver staining.
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