A variety
of PCR additives and enhancing agents have been used to increase the yield,
specificity and consistency of PCR reactions. Whilst these additives may have
beneficial effects on some amplifications it is
impossible to predict which agents will be useful in a particular context and
therefore they must be empirically tested for each combination of template and
primers. Some of the more popular of these additives are listed in the table
below along with references describing their use.
|
Additive |
References |
|
DMSO |
Amplifications 5: 16 |
|
Betaine |
Biochemistry 32: 137 |
|
Formamide |
Nucleic
Acids Research 18:
7465 |
|
Non-ionic
detergents |
Biotechniques 12: 332 |
|
TMAC |
Nucleic
Acids Research 18:
4953 |
|
7-deaza-2'-deoxyguanosine |
Nucleic
Acids Research 16:
3360 |
|
BSA |
Applied
and environmental microbiology 62:1102-1106 |
|
T4 gene 32 protein |
Applied
and Environmental Microbiology 62:1102-1106 |
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.(1998) 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 as a 5M solution from Sigma-Aldrich
(cat. # B 0300), 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 1989) 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.
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 1989).
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 (Proceedings of the National
Academy of Sciences of the United States of America 82: 1585) and
improve the stringency of hybridization reactions (Nucleic Acids Research
16: 4637).
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.
Frackman, S.,
Kobs, G., Simpson, D. and Storts, D. 1998. Betaine and DMSO: enhancing agents for PCR. Promega Notes 65: 27.
Gelfand, D. H. 1988. In
Erlich, H. A. (ed.) PCR Technology. p.17.
Stockton Press, NY.
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