PCR佐剂手册

 

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 (dimethyl sulfoxide)	Amplifications 5 : 16 Gene 140 : 1 Nucleic Acids Research 18 : 1666
Betaine (N,N,N-trimethylglycine = [carboxymethyl]trimethylammonium)	Biochemistry 32 : 137 BioTechniques 21 : 1102 Genome Research 6 : 633 Nucleic Acids Research 25 : 3957 Proceedings of the National Academy of Sciences of the United States of America 70 : 298 Trends in Biochemical Science 22 : 225
Formamide	Nucleic Acids Research 18 : 7465
Non-ionic detergents e.g. Triton X-100, Tween 20 or Nonidet P-40 (NP-40)	Biotechniques 12 : 332 Nucleic Acids Research 18 : 1309
TMAC (tetramethylammonium chloride)	Nucleic Acids Research 18 : 4953 Nucleic Acids Research 23 : 3343
7-deaza-2' -deoxyguanosine (dC7 GTP)	Nucleic Acids Research 16 : 3360
BSA (bovine serum albumin)	Applied and environmental microbiology 62 :1102-1106 BioTechniques 23 :504 BioTechniques 25 :564 Nucleic Acids Research 16 : 9775
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.

 

 

REFERENCES

 

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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