PCR additives

The article below was found over the internet. I have tried using DMSO with my PCR and it helps alot. I hope you guys find this helpfull. Do not waste your money on GC-rich buffer!


DMSO at 2-10% may be necessary for amplification of some templates, however 10% DMSO can reduce Taq polymerase activity by up to 50% (Gelfand 1989) so it should not be used routinely. DMSO is thought to reduce secondary structure and is particularly useful for GC rich templates.

A number of PCR additives are now comercially available, however the identities of these agents are not usually revealed by their suppliers. Frackman et al. (Promega Notes 65: 27) have demonstrated (using NMR analysis) that the PCR additive provided by QIAGEN in their PCR core kit (Q-Solution) and that provided by CLONTECH in the Advantage-GC cDNA PCR kit is in fact Betaine which is available at a fraction of the cost from Sigma-Aldrich, but be sure to use Betaine or Betaine (mono)hydrate and not Betaine HCl. Other products suspected of consisting largely of Betaine include the "GC-RICH solution enhancer" from Roche, "TaqMaster enhancer" from Eppendorf, "GC-melt" from Clontech and "FailSafe enhancer" (formerly "MasterAmp PCR Enhancment Technology") from Epicentre (Weissensteiner, pers. comm.). Betaine is generally used at a final concentration of 1.0-1.7M.

Formamide is generally used at 1-5% and 10% formamide is reported (Gelfand in Erlich (ed.), PCR Technology, p.17) to have no effect on the activity of Taq polymerase, however, Sarkar et al. (1990) (see table for ref.) found that 1.25% formamide worked as well as 2.5% and 5%, and no amplification was seen at 10% so it seems prudent not to use concentrations of formamide greater than strictly necessary for optimal amplification. For a disscusion of the potential use of other low molecular weight amides see Nucleic Acids Research 29: 2377-2381.

Non-ionic detergents stabilise Taq polymerase and may also supress the formation of secondary structure. 0.1-1% Triton X-100, Tween 20 or NP-40 may increase yield but may also increase non-specific amplification. As little as 0.01% SDS contamination of the template DNA (left-over from the extraction procedure) can inhibit PCR by reducing Taq polymerase activity to as low as 10%, however, inclusion of 0.5% Tween-20 or -40 will effectively neutralise this effect (Gelfand op. cit.).

TMAC is generally used at a final concentration of 15-100mM to eliminate non-specific priming. TMAC has is also used to reduce potential DNA-RNA mismatch (PNAS 82: 1585) and improve the stringency of hybridization reactions (Nucleic Acids Research 16: 4637). For a disscussion of the potential use of TMA oxalate see Nucleic Acids Research 28: e70.

The base analogue 7-deaza-2'-deoxyguanosine may facilitate amplification of templates with stable secondary structures when used in place of dGTP in a ratio of 3: 1, 7-deaza-2'-deoxyguanosine: dGTP.

BSA has proven particularly useful when attempting to amplify ancient DNA or templates which contain PCR inhibitors such as melanin.