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Using the TripleMaster PCR System for Robust Amplification of ,,, GC-Rich DNA Templates

Scott Tarpinian
Eppendorf 5 Prime, Boulder, CO, USA Introduction

The polymerase chain reaction (PCR*) is a powerful and sensitive technique used by scientists to amplify large quantities of DNA. Although the reaction mix and thermal cycling conditions can be adjusted to use almost any DNA sequence as a template, amplification of templates with high GC content has proven to be problematic. One difficulty is the bond stability between base pairs in GC-rich DNA sequences. Each G-C base pair contains three hydrogen bonds and is therefore more stable than an A-T base pair that has only two hydrogen bonds. Additional problems associated with the amplification of GC-rich DNA sequences during PCR are the renaturation of the template and the formation of secondary structures within each strand before the primers have a chance to anneal to their specific template sites. These properties of GC-rich DNA sequences lead to inefficient primer-template annealing and incomplete extension by the DNA Polymerase. As a result, amplification often results in little or no yield of the expected product when using standard PCR methods. In addition, amplification may result in non-specific products derived from regions other than the target DNA sequence.1, 3, 4, 6

The TripleMaster PCR System's unique mixture of three different enzymes (Taq DNA Polymerase, 3'5'proofreading enzyme, and polymerase enhancing factor) combined with its innovative reaction buffers, overcomes these obstacles and provides scientists with an ideal solution for amplification of complex GC-rich DNA templates.5

Materials

Eppendorf TripleMaster PCR System
Invitrogen ThermalAce DNA Polymerase Kit
Roche GC-RICH PCR System
Genomic DNA isolated using the Eppendorf Perfect gDNA Blood Mini Kit
Eppendorf Mastercycler Gradient Thermal Cycler
EasyCast Horizontal Minigel System (Owl Separation Systems)
SeaKem LE Agarose (BioWhittaker Molecular Applications)
1x TBE (Eppendorf)
Ethidium Bromide (Sigma)
Alpha Imager CCD Camera Documentation System (Alpha Innotech Corporation)


Methods

The performance of the TripleMaster PCR System for GC-rich PCR was tested against other PCR systems specifically designed for GC-rich PCR. Three different DNA sequences with high GC-content were chosen for amplification. The primer sequences, length of the expected PCR products and percentage of GC content in the amplified sequences are listed in Table 1. Genomic DNA was isolated from whole blood preserved in EDTA using the Eppendorf Perfect gDNA Blood Mini Kit. The oligonucleotide primers were obtained from Sigma-Genosys. Triplicate reactions were prepared in accordance with the manufacturers recommended specifications and all PCR reagents were used at final concentrations within the ranges recommended by the manufacturer (see Table 2). The 1.6x Eppendorf Tuning Buffer concentration (with 4.0 mM Mg2+) was used for the apolipoprotein E and retinoblastoma gene reactions due to the size of the targets. The TripleMaster PCR System user manual recommends a higher buffer concentration for targets less than 500 bp because the Tuning Buffer is a low-salt buffer specifically designed for long-range PCR, and short targets require a buffer with a higher salt concentration. The GC-RICH Resolution Solution of the Roche system was tested at concentrations of 0 2 M in 0.5 M increments, as stated in the user manual. All amplifications were performed on an Eppendorf Mastercycler Gradient (see Table 3). Samples were analyzed by standard agarose gel electrophoresis in a 1x TBE buffer. 5 ml of the 50 ml reactions were loaded on a 2.0% agarose gel, run at 6 V/cm, stained in a 0.5 mg/ml ethidium bromide solution and photographed with a CCD camera.

In experiment 1, the TripleMaster PCR System was used to determine the optimal reaction mix and thermal cycling conditions for the apolipoprotein E and retinoblastoma targets. Reactions were then set up in parallel to compare the TripleMaster PCR System with the leading suppliers.

In experiment 2, the TripleMaster PCR System was used to determine the reaction mix conditions that generated the highest product yield for the MC1R target using standard thermal cycling parameters (95C for 5 minutes, followed by 35 cycles at 95C for 30 seconds, 56C for 30 seconds, and 72C for 1 minute). Touchdown PCR was then employed to prove this method is capable of achieving optimal amplification and specificity without the need for thermal cycling optimization experiments. Reactions were set up in parallel to compare the performance of the TripleMaster PCR System with the performance of the leading suppliers.


Table 1: Primer sequences, amplicon size and GC content for GC-rich PCR targets.

Target Amplicon % GC Primer Sequences Human retinoblastoma gene (RB) 180 bp 75% 5' CAG GAC AGC GGC CCG GAG 3'
5 CTG CAG ACG CTC CGC CGT 3' Human apolipoprotein E gene (apo E) 268 bp 75% 5' GGC ACG GCT GTC CAA GGA 3'
5' CTC GCG GAT GGC GCT GAG 3' Human melanocortin-1 receptor gene (MC1R) 760 bp 63% 5' CTG GTG AGC TTG GTG GAG A 3'
5' GGC TGT GGA AGG CGT AGA T 3' Table 2: Final reagent concentrations in 50 ml reaction volumes. Eppendorf TripleMaster PCR System Human RB gene(180 bp) Human apo E gene (268 bp) Human MC1R gene (760 bp) Tuning Buffer with Mg2+ 1.6x; 4.0 mM 1.6x; 4.0 mM 1x; 2.5 mM DMSO 5% 5% 2% dNTP's 200 M each 200 M each 200 M each Primers 0.4 M each 0.4 M each 0.4 M each TripleMaster enzyme mix 2.0 U 2.0 U 2.0 U Template DNA 100 ng 100 ng 100 ng Invitrogen ThermalAce DNA Polymerase Kit Human RB gene(180 bp) Human apo E gene (268 bp) Human MC1R gene (760 bp) ThermalAce Buffer with Mg2+ 1x; 1.5 mM 1x; 1.5 mM 1x; 1.5 mM dNTP's 200 M each 200 M each 200 M each Primers 0.4 M each 0.4 M each 0.4 M each ThermalAce DNA Polymerase 2.0 U 2.0 U 2.0 U Template DNA 100 ng 100 ng 100 ng Roche GC-RICH PCR System Human RB gene(180 bp) Human apo E gene (268 bp) Human MC1R gene (760 bp) GC-RICH PCR Buffer with Mg2+ and DMSO 1x; 1.5 mM; 5% 1x; 1.5 mM; 5% 1x; 1.5 mM; 5% GC-RICH Resolution Solution 0 2 M 0 2 M 0 2 M dNTP's 200 M each 200 M each 200 M each Primers 0.4 M each 0.4 M each 0.4 M each GC-RICH PCR Enzyme mix 2.0 U 2.0 U 2.0 U Template DNA 100 ng 100 ng 100 ng Table 3: Thermal cycling parameters for GC-rich PCR targets. Target Cycle Temperature Time Human RB gene (180 bp) 1x 95C 5 min. 35x 95C 20 sec. 35x 69C 10 sec. 35x 72C 30 sec. Human apo E gene (268 bp) 1x 95C 5 min. 35x 95C 20 sec. 35x 60C 10 sec. 35x 72C 30 sec. Human MC1R gene (760 bp) 1x 95C 5 min. 25x 94C 1 min. 25x 63C with 0.5C/cycle decrease 1 min. 25x 72C 1 min. 30 sec. 10x 94C 1 min. 10x 50C 1 min. 10x 72C 1 min. 1x 72C 10 min. Results Experiment 1

All three PCR systems amplified the 268 bp apo E gene target, with TripleMaster producing the highest yield (see Fig. 1). The Invitrogen and Roche systems produced approximately equal yields, with Roche amplifying the desired product without GC-RICH Resolution Solution and with 0.5 M GC-RICH Resolution Solution. Roche did not amplify any products with GC-RICH Resolution Solution concentrations of 1.0 M, 1.5 M, or 2.0 M (data not shown).

The TripleMaster and Invitrogen systems amplified the 180 bp RB gene target with approximately equal yield (see Fig. 1). However, the Invitrogen system also amplified non-specific products. The Roche system did not amplify any PCR products for all concentrations of the GC-RICH Resolution Solution (data not shown).

Fig. 1: The TripleMaster PCR System compared to leading suppliers in the amplification of GC-rich templates from the human apo E and RB genes.
Lanes:

M New England Biolabs 100 bp Marker (0.5 g) 1 Eppendorf, apo E gene 2 Eppendorf, RB gene 3 Invitrogen, apo E gene 4 Invitrogen, RB gene 5 Roche, apo E gene, without GC-RICH Resolution Solution 6 Roche, apo E gene, with 0.5 M GC-RICH Resolution Solution
Results Experiment 2

The initial thermal cycling conditions used for the MC1R target with the TripleMaster system amplified the desired 760 bp MC1R gene target with excellent yield, but also amplified multiple non-specific products (see Fig. 2). However when the touchdown PCR cycling protocol was used, all three systems amplified only the desired target with outstanding yield. The TripleMaster system amplified the most robust product, with the Invitrogen and Roche systems producing slightly less yield. Roche amplified the MC1R target without GC-RICH Resolution Solution and with GC-RICH Resolution Solution concentrations of 0.5 M and 1.0 M. Roche did not amplify any products with concentrations of 1.5 M or 2.0 M (data not shown).

Fig. 2: The TripleMaster PCR System compared to leading suppliers in the amplification of a GC-rich template from the human MC1R gene.
Lanes:


M New England Biolabs 100 bp Marker (0.5 g) 1 Eppendorf, standard thermal cycling protocol 2 Eppendorf, touchdown PCR protocol 3 Invitrogen, touchdown PCR protocol 4 Roche, touchdown PCR protocol, without GC-RICH Resolution Solution 5 Roche, touchdown PCR protocol, with 0.5 M GC-RICH Resolution Solution 6 Roche, touchdown PCR protocol, with 1.0 M GC-RICH Resolution Solution
Discussion

The TripleMaster system required the use of DMSO (dimethyl sulfoxide) to improve the yield and specificity of the PCR reactions for these specific GC-rich templates. DMSO is a co-solvent that destabilizes DNA by disrupting base pairs and thereby lowering the melting temperature of DNA. DMSO has been known to improve the amplification of some GC-rich DNA sequences, including some resistant to amplification by standard PCR techniques. While DMSO improved the yield and specificity of amplification for these targets, it may not be beneficial or necessary for other targets. In these cases, other PCR additives such as betaine, formamide, glycerol or trimethylammonium hydrochloride can be tested. However it is impossible to identify which, if any, PCR additive is best for a particular target, so it may be necessary to test several different agents separately or in combination. It should be noted that although these additives can increase the yield and specificity of PCR reactions, their effects on the melting temperature of DNA alters the optimal annealing temperature for a specific primer/template system. Therefore, optimization of the thermal cycling parameters may be required, which often requires the performance of several experiments.1, 3, 4, 5, 6

One method that eliminates the need for thermal cycling optimization experiments is the touchdown PCR method, which is used in the amplification of the MC1R target. During touchdown PCR, the annealing temperature of the first cycle is set at or a few degrees above the melting temperatures of the primers. The annealing temperature of each sequential cycle is then programmed for incrementally lower temperatures so that the annealing temperature range of the entire program spans approximately 15C. This favors amplification of the target amplicon by ensuring that the first annealing event involves the template sequence with the greatest complement to the primers. Although the annealing temperature eventually drops to a level where non-specific products are normally formed, the target amplicon has already commenced its exponential amplification and therefore surpases any non-specific products during these cycles. Touchdown PCR is a valuable method for generating robust PCR reactions without time-consuming optimization experiments.2

In conclusion, the TripleMaster Enzyme Mix combines the efficiency of Taq DNA Polymerase with the 3' 5' exonuclease activity of a proofreading thermostable polymerase, along with a polymerase-enhancing factor that provides an extremely high extension rate and maximal proofreading assisted fidelity. The TripleMaster Tuning Buffer, specifically designed for long-range PCR, can also be used for the amplification of short, GC-rich targets. In addition, the TripleMaster High Fidelity Buffer can be used for high-fidelity PCR as well as the amplification of short, GC-rich targets (data not shown). All of these innovative components make the TripleMaster PCR System a versatile kit that can be used for the amplification of GC-rich targets, long-range PCR, and high-fidelity PCR. One can see how the TripleMaster PCR System is a universal solution for a variety of PCR applications.5

References:

  1. Liu, Q. and S.S. Sommer (1998) Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene. Biotechniques. 25: 10221028.
  2. Roux, K.H. (1995) Optimization and Troubleshooting in PCR. In: PCR Primer, A Laboratory Manual (Dieffenback, C.W. and G.S. Dveksler, editors), Cold Spring Harbor Laboratory Press, NY, pp. 5362.
  3. Schuchard, M., G. Sarkar, T. Ruesink and T.C. Spelsberg (1993) Two-step hot PCR amplification of GC-rich avian c-myc sequences. Biotechniques. 14: 390394.
  4. Sun, Y., G. Hegamyer and N.H. Colburn (1993) PCR-direct sequencing of a GC-rich region by inclusion of 10% DMSO: application to mouse c-jun. Biotechniques. 15: 372374.
  5. TripleMaster PCR System user manual (2000) Version TM1000, Eppendorf.
  6. Turner, S.L. and F.J. Jenkins (1995) Use of deoxyinosine in PCR to improve amplification of GC-rich DNA. Biotechniques. 19: 4852.
* The Polymerase Chain Reaction (PCR) is covered by U.S. patent numbers 4683105 and 4683202 held by Roche Molecular Systems and requires a license for use.


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