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Augmented PCR amplification by thermally activated DNA polymerase - AmpliTaq Gold, in the presence of densifying agents.

Duraiswamy Navaneetham* and Bianca M. Conti-Fine

AmpliTaq Gold is a chemically modified form of AmpliTaq, a DNA polymerase commonly used in various polymerase chain reaction (PCR) protocols. We identified that the activity of AmpliTaq Gold DNA polymerase is influenced by densifying agents like sucrose and glycerol. This property of the enzyme was analyzed at 0-24% sucrose and glycerol concentrations, and found that concentrations between 12% and 18% had positive influence, in terms of PCR product intensity on agarose gel. Therefore, inclusion of sucrose or glycerol in PCR mixture would increase PCR product band intensity when AmpliTaq Gold is employed. Cresol red, a dye is also found to be compatible with AmpliTaq Gold. Both densifying agents and cresol red eliminate the need for post-PCR manipulation in order to load the reaction mixture onto the electrophoretic gel. 

Various DNA polymerases are used to amplify target DNA sequences. The amplification specificity can be increased by the use of manual ‘hot start’ polymerase chain reaction (PCR) protocols (1-2,4) by using materials like paraffin wax (1-2), petroleum jelly (4) etc., to separate from the reaction mixture one or more essential components, like DNA polymerase, primer or template.  The missing component(s) is allowed into the PCR mixture once a ‘favorable’ temperature is reached, that solubilized the separating septum. 

DNA polymerases that can be thermally activated are commercially available (AmpliTaq Gold, a chemically modified form of AmpliTaq DNA polymerase, [Perkin-Elmer, Norwalk, CT] and monoclonal antibody tagged PLATINUM^TM Taq [Life Technologies, Inc., Gaithersburg, MD]).  These enzymes are inactive at room temperature and are activated upon heating.  Their use had simplified the protocol by eliminating manual ‘hot start’ procedures. 

PCR protocols can be improved also by adding to the PCR mixture a PCR compatible dye, cresol red and an inert densifying agent, sucrose (3).  This allows to visualize if all components were added while setting up the PCR samples, and to direct transfer of the PCR products onto the gel without any post-PCR manipulations (3).  Cresol red also serves as a marker dye during the gel electrophoresis of the PCR products (3).  We have been routinely and successfully using AmpliTaq in the presence of sucrose and cresol red, in manual ‘hot start’ protocols (5). To further optimize the advantages of thermally activated DNA polymerase, we have determined whether densifying materials, such as sucrose or glycerol, and cresol red are compatible with the use of a thermally activated DNA polymerase.  Here we describe the influence of these agents on AmpliTaq Gold.

We used template cDNAs from the Jurkat human T cell line (American Type Culture Collection, Rockville, MD). Total RNAs were prepared by homogenizing 10x10⁶ cells in phenol-guanidinium thiocyanate based RNAsol B or RNA STAT 60 (Tel-Test Inc., Friendswood, TX).  After precipitation and washing, the total RNA pellets were dissolved in 10 ml of H₂O treated with 0.02% diethyl pyrocarbonate (Sigma Chemical Co. St. Louis, MO).  Twenty ml reactions were set up for reverse transcription (RT) using 2 ml of RNA, and Moloney murine leukemia virus reverse transcriptase (SuperScript RNase H^-; Gibco-BRL, Gaithersburg, MD).  After the RT reactions the volumes of the mixtures were adjusted to 100 ml with H₂O.  PCR amplification was carried out using the reaction buffer II (Perkin-Elmer, Norwalk, CT), 200 mM dNTPs, 2 mM MgCl₂, 0.5 mM primers, varying concentrations of sucrose or glycerol, 0.5 U AmpliTaq Gold DNA polymerase and 1 ml of cDNA, in a total volume of 10 ml.  PCRs were performed in a GeneAmp PCR System 9600 (Perkin-Elmer) using the following cycling parameters: activation for 12 min. at 95^oC followed by 30 cycles at 94^oC s for 30 s, 55^oC for 30 s and 72^oC for 60 s.  The PCR products were resolved on 1% agarose gel containing ethidium bromide (0.1mg/100ml).  The primers we used were: 5'-aggatcttcatgaggtagt-3' and 5'-gctccggcatgtgcaa-3' (actin forward and reverse primers, respectively).  We prepared sucrose (Life Technologies, Inc., Gaithersburg, MD) and glycerol (Fisher Scientific, Fair Lawn, NJ) as 60% stock solutions in H₂O that contained 1 mM cresol red (Aldrich Chemical Company, Milwaukee, WI). 

The presence of increasing amounts (12-18%) of sucrose increased the yield of PCR products for actin (Figure 1A), using AmpliTaq Gold DNA polymerase.  Glycerol had the same effect (Figure 1B).  The sucrose stock solution contained 1 mM cresol red:  therefore the PCR reactions occurred in the presence of up to 0.4 mM cresol red. We analyzed if cresol red had any role in increased PCR product yield. Concentrations of cresol red up to 0.8 mM did not affect the yield of the PCR products (Figure 1C).  Concentrations of sucrose and glycerol above 18% appeared to be inhibitory.  The inhibitory effects were more pronounced for glycerol than sucrose (Figure 1A & B).

Figure 1. Influence of sucrose, glycerol and cresol red on AmpliTaq Gold.

(A) Actin PCR amplification products (542 bp) in the presence of 0-24% sucrose using Jurkat cDNA (lanes 1-6; 30 cycles) and 100 bp ladder (lane 7; Life Technologies, Gaithersburg, MD).

(B) Actin (542 bp) amplification using Jurkat cDNA in the presence of 0-24% glycerol (lane 1-6; 30 cycles).

(C) Actin (542 bp) amplification in the presence of 6% sucrose and varying concentration of cresol red using Jurkat cDNA (lanes 1-6; 30 cycles).
 

This study demonstrates the use of ‘inert’ densifying agents such as sucrose and glycerol that increased the yield of PCR products obtained using a thermally activated DNA polymerase. The increase in PCR product yield when using a densifying agent is a property of the AmpliTaq Gold, because the presence of sucrose has no effect on the product  yield of the unmodified AmpliTaq DNA polymerase (data not shown). 

The inclusion of 12-18% sucrose or glycerol in the PCR mixture has the dual advantage of increasing the product yield and facilitating the direct transfer of PCR products onto the gel.  The increased yield of the PCR products by using a densifying agent allows the use of less template for PCR amplification.  The number of PCR cycles necessary to achieve a satisfactory quantity of amplified products may also be reduced.  Finally, the presence in the PCR reaction mixture of a densifying agent and a dye eliminates the post-PCR manipulations needed to add this sort of agents prior to loading the products onto the gel. Thus handling large number of PCR samples will be easier and faster. 

This study for the first time reports the beneficial influence of sucrose and glycerol on AmpliTaq Gold, and the compatibility of cresol red with this enzyme. Further studies are needed to ascertain the influence of these agents in the fidelity of DNA duplication, and the molecular mechanism behind the augmented PCR amplification of the AmpliTaq Gold.

1. Bassam, B.J. and G. Caetano-Anolles. 1993. Automated ‘hot start’ PCR using mineral oil and paraffin wax. BioTechniques 14: 31-33. 

2. Chou, Q., M.  Russell, D.E. Birch, J. Raymond and W. Bloch 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Research  20: 1717-1723. 

3. Hoppe, B.L., B.M. Conti-Tronconi and R.M. Horton. 1992. Gel-loading dyes compatible with PCR.  BioTechniques  12: 679-680. 

4. Horton, R.M., B.L. Hoppe and B.M.  Conti-Tronconi. 1994.  AmpliGrease: "Hot Start" PCR using petroleum jelly. BioTechniques  16: 42-43. 

5. Navaneetham D., A. Penn,  J. Howard, Jr. and B.M. Conti-Fine.  1997. Expression of the a7 subunit of the nicotinic acetylcholine receptor in normal and myasthenic human thymuses. Cellular and  Molecular Biology  43: 433-442. 

© 1998-2001 Duraiswamy Navaneetham