We're trying to do emulsion PCR using HA-coated polystyrene beads and we're noticing that the beads are seeing drastic issues with thermal degradation above 90C. As PCR has an unfortunate requirement of requiring a high temperature, I was curious about what options are out there to reduce the temperature during PCR denaturation or to stabilize the beads.
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1$\begingroup$ Unfortunately melting temperature of nucleic acid is an intrinsic property of the molecule. Do you need to use polystyrene as a substrate? Perhaps silicon or glass are better suited for this in situ PCR. $\endgroup$– user560Commented Nov 28, 2012 at 2:58
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$\begingroup$ Well you can change the melting temperature using additives like DMSO and betaine. Unfortunately, the beads have to be coated and the polystyrene seems to be the best for the downstream steps so we would prefer to stick with polystyrene before re-optimizing everything else. $\endgroup$– bobthejoeCommented Nov 28, 2012 at 6:18
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2$\begingroup$ That's true, but there is a diminishing return on additives (at least DMSO) in which after a certain threshold either the polymerase will no longer function or the reaction will shift towards non-specific priming. $\endgroup$– user560Commented Nov 28, 2012 at 15:39
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$\begingroup$ If there was an easy solution, I wouldn't be asking it on SE. $\endgroup$– bobthejoeCommented Nov 28, 2012 at 18:51
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$\begingroup$ I've seen PCR melt temps as low as 92 C. It's too low a temp to melt, but do they become fluid > 90 C? $\endgroup$– user560Commented Nov 28, 2012 at 23:08
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3 Answers
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Perhaps isothermal amplification is possible (NASBA)?
Amplification of DNA also seems possible (NASBA at biomerieux).
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Look at chemical hydrogen bond breakers. Guanadine hydrochloride is included in the buffer conventionally to compete with the the hydrogen bonds that form the double helix. Other hydrogen bonders like Propionamide and 2-Pyrrolidone can weaken the helix and I would think lower the melting point of the bonds.
How much the Tm is affected would depend on the concentration of the breaker in the buffer. It might not be possible to lower the temperature this much and you may loose specificity - you might be able to squeeze a few more degrees if the oligos can't get any shorter.
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$\begingroup$ Doesn't this accomplish the same thing as DMSO? $\endgroup$– user560Commented Dec 6, 2012 at 13:13
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$\begingroup$ i think a few molar salt will also do it, or just amping up the Gu in the solution, but the paper also tries to estimate specificity and fidelity of the PCR reaction with different compounds so that you don't get as much error with increased bond breaking. In the extreme concentrations of adding bond breakers, you get no double helix formation at all. $\endgroup$– shigetaCommented Dec 6, 2012 at 17:40
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Update (21/09/17) as the answers are a little outdated by now:
Isothermal amplification methods are fairly well established by now and commercially available. They tend to amplify DNA at temperatures of around 56°C.
- Wikipedia entry on Loop-mediated isothermal amplification.
- For the more tech-savvy, an open research article on loop-mediated isothermal amplification in Nucleic Acids Research.
- Link to a commercially available product using isothermal amplification: ANSR (Disclaimer: I work for this company)
