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TripleMaster PCR SystemThe Ideal Choice for Long-Range PCR

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

PCR* technology has been widely used in molecular biology research, providing scientists with an efficient tool for studies such as genome analysis, sequencing, and PCR cloning for gene expression. However, under standard PCR conditions, amplification of DNA fragments greater than 5 kb has been problematic. One major factor contributing to the inability of standard PCR to amplify long segments of DNA is template degradation during exposure to high temperatures in buffers that are unable to maintain pH. This is due to depurination, the hydrolysis of the N-glycosidic bond between the base and the sugar of a DNA molecule. Although depurination is a rare event, occurring approximately once every 25 kb of single-stranded DNA per minute, its effect on long-range PCR is more profound because long templates simply have more sites that are susceptible to depurination. In addition, depurination is more prevalent at the higher temperatures and lower pHs necessary for amplification of long targets. And once the N-glycosidic bond is cleaved, Taq Polymerase is unable to extend past the site of depurination and amplification of the desired target is not possible. Another crucial factor in the limitation of standard PCR to amplify long target sequences is the high rate of incorporation of incorrect bases by thermostable polymerases, such as Taq DNA Polymerase, that lack an editing function. The Eppendorf TripleMaster PCR System solves both of these problems with its innovative buffer system and unique enzyme mix, making it the ideal choice for scientists seeking to overcome the limitations of standard PCR systems.1, 2, 3, 4

The TripleMaster PCR Systems Tuning Buffer has a unique zwitterionic formulation (patent pending) that adequately maintains pH at high temperatures (72 0C 94 0C), reducing template and amplicon degradation which is caused by depurination to a minimum. In addition, the TripleMaster Enzyme Mix combines the efficiency of Taq DNA Polymerase with the 3 5 exonuclease activity of a proofreading thermostable enzyme, along with a polymerase-enhancing factor that provides an extremely high extension rate and maximal proofreading assisted fidelity.5

Materials and Methods

Eppendorf TripleMaster PCR System
Invitrogen ThermalAceTM DNA Polymerase Kit
Stratagene EXLTM DNA Polymerase Kit
Roche Expand Long Template PCR System
Roche Expand 20 kbPlus PCR System
Genomic DNA isolated using the Eppendorf Perfect gDNA Blood Mini Kit
Eppendorf Mastercycler Gradient Thermal Cycler
FIGE Mapper Electrophoresis System (Bio-Rad)
OmniPur Agarose (EM Science)
0.5x TBE (Eppendorf)
Ethidium Bromide (Sigma)

The performance of the TripleMaster PCR System for long-range PCR was tested against other PCR systems specifically designed for long-range PCR. A 29 kb fragment of the human Factor VIII gene and 24 kb and 18 kb fragments of the human tPA gene were amplified in parallel reactions. All reactions were treated and set up in accordance with the manufacturers recommended specifications. For the Roche systems, the Expand 20 kbPlus PCR System was used to amplify the 29 kb and 24 kb targets and the Expand Long Template PCR System was used to amplify the 18 kb target. Primers specific to the human Factor VIII gene and the human tPA gene were synthesized by Integrated DNA Technologies or Sigma-Genosys (see Table 1). All PCR reagents were used at the final concentrations recommended by the manufacturer (see Table 2). Genomic DNA was isolated from whole blood preserved in EDTA using the Eppendorf Perfect gDNA Blood Mini Kit. For all instances in which a competitors PCR system did not contain a necessary reagent, such as molecular biology grade water or dNTP's, the Eppendorf version of that reagent was used. All thermal cycling was performed on an Eppendorf Mastercycler Gradient using optimized parameters for the specific primer/template system (see Table 3). Samples were analyzed by field inversion gel electrophoresis in a 0.5x TBE buffer using a 180 V forward voltage, a 120 V reverse voltage, and a switch time ramp of 0.1 0.8 seconds (linear shape). 15 ml of the 50 ml reactions were loaded on a 1.0% agarose gel, run for 16 hours, stained in a 0.5 mg/ml ethidium bromide solution and photographed.

Table 1: Primers sequences for long-range PCR targets.

Primer Sequence Factor VIII Forward 5' ACC CAC CAG TCT TGA AAC GC 3' Factor VIII Reverse 5' GGT GCT CCA GGC ATT GAT TG 3' tPA Forward 5' CCT TCA CTG TCT GCC TAA CTC CTT CGT GTG TTC C 3' tPA 24 kb Reverse 5' TGT CTC CAG CAC ACA GCA TGT TGT CGG TGA C 3' tPA 18 kb Reverse 5' GCA GGG TCT GCA GAA CTC TGA GCT GTA CTT CC 3' Table 2: Final reagent concentrations for long-range PCR reactions. Eppendorf TripleMaster Human Factor VIII gene 29 kb Human tPA gene 24 kb Human tPA gene 18 kb Tuning Buffer 1x 1x 1x Magnesium 2.5 mM 2.5 mM 2.5 mM dNTP's 500 M 500 M 500 M Forward primer 0.4 M 0.4 M 0.4 M Reverse primer 0.4 M 0.4 M 0.4 M TripleMaster enzyme mix 2 U 2 U 2 U Template DNA 500 ng 500 ng 500 ng Invitrogen ThermalAce Human Factor VIII gene 29 kb Human tPA gene 24 kb Human tPA gene 18 kb ThermalAce Buffer 1x 1x 1x Magnesium 1.5 mM 1.5 mM 1.5 mM dNTP's 200 mM @@200 M 200 mM @@200 M 200 mM @@200 M Forward primer 100 ng 100 ng 100 ng Reverse primer 100 ng 100 ng 100 ng ThermalAce DNA Polymerase 2 U 2 U 2 U Template DNA 100 ng 100 ng 100 ng Stratagene EXL Human Factor VIII gene 29 kb Human tPA gene 24 kb Human tPA gene 18 kb EXL Buffer 1x 1x 1x Stabilizing solution 1 l 1 l 1 l Magnesium Not listed Not listed Not listed dNTP's 500 M 500 M 500 M Forward primer 200 ng 200 ng 200 ng Reverse primer 200 ng 200 ng 200 ng EXL DNA Polymerase 5 U 5 U 5 U Template DNA 250 ng 250 ng 250 ng Roche Expand 20 kbPlus & Expand Long Human Factor VIII gene 29 kb Human tPA gene 24 kb Human tPA gene 18 kb Buffer 1x 1x 1x Magnesium 2.25 mM 2.25 mM 2.25 mM dNTP's 500 M 500 M 500 M Forward primer 0.4 M 0.4 M 0.3 M Reverse primer 0.4 M 0.4 M 0.3 M Enzyme mix 2.6 U 2.6 U 2.6 U Template DNA 250 ng 250 ng 250 ng Table 3: Thermal cycling parameters for long-range PCR targets. Target Cycle Temperature Time 29 kb Human Factor VIII gene 1x 93C 3 min. 10x 93C 15 sec. 10x 57C 30 sec. 10x 68C 24 min. 18x 93C 15 sec. 18x 57C 30 sec. 18x 68C 24 min. 20 sec. 24 kb Human tPA gene 1x 93C 3 min. 10x 93C 15 sec. 10x 65C 30 sec. 10x 68C 21 min. 17x 93C 15 sec. 17x 65C 30 sec. 17x 68C 21 min. 20 sec. 18 kb Human tPA gene 1x 93C 3 min. 10x 93C 15 sec. 10x 65C 30 sec. 10x 68C 15 min. 17x 93C 15 sec. 17x 65C 30 sec. 17x 68C 15 min. 20 sec. Results

The Eppendorf TripleMaster PCR System and the Roche PCR Systems amplified all three long-range PCR targets, whereas the Invitrogen and Stratagene systems did not amplify any targets (see Fig. 1). In addition, the Eppendorf TripleMaster PCR System amplified PCR products with a yield superior to the Roche PCR Systems. The U-shaped bands observed in some samples are due to the use of a glycerol-based loading buffer that allowed some of the DNA to stream up the sides of the wells during electrophoresis. Furthermore, due to the equal amount of reaction volume loaded on the gel for each competitors samples, the Eppendorf samples were overloaded, resulting in trailing and smearing. This problem was accentuated by the large size of the DNA fragments.

Fig. 1: Long-range PCR results comparing the TripleMaster PCR System to leading suppliers.
Lanes:

M Bio-Rad 8 kb 48 kb marker 13 Eppendorf reactions 46 Invitrogen reactions 79 Roche reactions 1012 IStratagene reactions Discussion

The TripleMaster PCR System amplified the most robust PCR products in all cases, easily outperforming the competition. Roche, the closest competitor, has one system for amplification of targets 5 kb 20 kb in size and another system for amplification of targets greater than 20 kb. However, the Eppendorf TripleMaster PCR System can amplify targets across this entire range so scientists only need to purchase one system for all long-range PCR applications. This enables researchers to save valuable time and money when ordering a PCR system for long-range PCR. Furthermore, the TripleMaster PCR System contains a straightforward, easy to use protocol that recommends specific reagent concentrations instead of a broad range of concentrations, avoiding the need for time-consuming optimization experiments. The TripleMaster System is also designed to amplify long targets up to 50 kb without the need for co-solvents or additives such as betaine or DMSO, further simplifying the master mix set-up. In addition, compared with all the systems tested, the TripleMaster PCR System used the least amount of enzyme per reaction, proving that it not only generates PCR products with the greatest yield, it is also the most cost-effective system for long-range PCR. One can see that the Eppendorf TripleMaster PCR System is the ideal choice for long-range PCR.5

References:

  1. Barnes, W.M. (1994) PCR amplification of up to 35 kb DNA with high fidelity and high yield from l bacteriophage templates. Proc. Natl. Acad. Sci. USA. 91: 2216-2220.
  2. Cantor, C.R. and C.L. Smith (1999) Genomics. John Wiley & Sons, Inc., NY, pp. 106107.
  3. Cheng, S., C. Fockler, W.M. Barnes and R. Higuchi (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA. 91: 56955699.
  4. Frost, M.R. and J.A. Guggenheim (1999) Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels. Nucleic Acids Res. 15: e6.
  5. TripleMaster PCR System user manual (2000) Version TM1000, Eppendorf.
* 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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