PCR Additives

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.

http://www.staff.uni-mainz.de/lieb/additiva.html

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 America70: 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 (dC⁷GTP)	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.

A new PT scheme has been developed, providing an opportunity for users to obtain confidential and unbiased assessment of their QPCR performance. The PT has been developed under the DTI-funded Measurements for Biotechnology (MfB) programme as part of an initiative to improve the comparability of results between laboratories, and the materials for the first round will be sent out this September.
The scheme takes an innovative approach, and uses synthetic DNA targets in a proprietary artificial matrix to allow researchers and analysts from all sectors to participate without fears of laboratory contamination. Participants will be required to perform basic DNA quantification of a high concentration DNA stock, DNA extraction of 9 unknown samples and QPCR analysis of the 9 unknown samples plus 3 additional unknowns. Results will be compared between participating laboratories, and performance will be scored using a conventional PT Z-scoring approach. By taking part in the scheme, participants will be able to demonstrate the overall effectiveness of their performance, which is increasingly important to maintain the confidence of customers and funding bodies.

EQUAL-quant: an international external quality assessment scheme for real-time PCR.

Ramsden SC, Daly S, Geilenkeuser WJ, Duncan G, Hermitte F, Marubini E, Neumaier
M, Orlando C, Palicka V, Paradiso A, Pazzagli M, Pizzamiglio S, Verderio P.
Clin Chem. 2006 Aug;52(8):1584-91

National Genetics Reference Laboratory (Manchester), St. Mary's Hospital,
Manchester, UK, and Institute of Medical Statistics and Biometry, University of Milan, Italy.

Diagnostic PCR: validation and sample preparation are two sides of the same coin.

Hoorfar J, Wolffs P, Radstrom P.
APMIS. 2004 Nov-Dec;112(11-12):808-14.

Department of Microbiological Food Safety, Danish Institute for Food and
Veterinary Research (DFVF), Copenhagen, Denmark.

Evaluation of a Homemade SYBR® Green I Reaction Mixture for Real-Time PCR Quantification of Gene Expression.
Albert Karsai, Sabine Müller, Stefan Platz, and Marie-Theres Hauser (2002)
University of Agricultural, Sciences Vienna, Austria

Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBR® Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result was obtained with cDNAs synthesized by OmniScript reverse transcriptase from 2.5 µg total RNA using oligo d(T)18 primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the reproducibility and amplification efficiency was high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed us to quantify 10–20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.

Rapid, single-tube method for quantitative preparation and analysis of RNA and
DNA in samples as small as one cell.

Hartshorn C, Anshelevich A, Wangh LJ.
BMC Biotechnol. 2005 Jan 13;5(1):2.

Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.

Potential influence of the first PCR cycles in real-time comparative gene quantifications.

Nogva HK, Rudi K.
Biotechniques. 2004 Aug;37(2):246-8, 250-3. 



Norwegian Food Research Institute, AS, Norway.

There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.

Reagent volume and plate bias in real-time polymerase chain reaction.

Thomson E, Vincent R.
Anal Biochem. 2005 Feb 15;337(2):347-50. 



Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ont.,
Canada K1A 0L2.

When optimal conditions are employed, real-time polymerase chain reaction (PCR) is a sensitive and accurate technique enabling the quantiWcation of low-copynumber transcripts. However, as with conventional PCR, small variations in initial reaction conditions are amplified exponentially and can signiWcantly aVect results. Uniform reaction conditions are therefore essential to achieve accuracy and reproducibility during transcript quantiWcation. Reagent costs are considerable, and in a high-throughput setting a reduction in the reagent volume used in each reaction would signiWcantly reduce the cost of real-time PCR. However, the eVect of reduced volume on the accuracy of results in a plate-based system has not, to our knowledge, been examined. Here we show that lower reagent volumes can reduce reproducibility by enhancing a bias in results across a plate.