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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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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. – leonardo Nov 28 '12 at 2:58
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. – bobthejoe Nov 28 '12 at 6:18
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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. – leonardo Nov 28 '12 at 15:39
If there was an easy solution, I wouldn't be asking it on SE. – bobthejoe Nov 28 '12 at 18:51
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? – leonardo Nov 28 '12 at 23:08

2 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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Doesn't this accomplish the same thing as DMSO? – leonardo Dec 6 '12 at 13:13
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. – shigeta Dec 6 '12 at 17:40

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