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High-Fidelity PCR with a Novel Polymerase Mixture

New polymerase mixture, TaqPlus Precision PCR system, developed for highfidelity PCR


Kirk B. Nielson * Janice Cline * Frances Bai * Daniel McMullan * Barbara McGowan * Holly Hogrefe
Stratagene Cloning Systems, Inc.


Stratagene announces the addition of the TaqPlus Precision PCR system ,* to an expanding line of PCR products. The TaqPlus Precision PCR system consists of a novel mixture of Taq2000 and Pfu DNA polymerases and a reaction buffer that has been developed for high-fidelity PCR applications. As with many two-enzyme PCR mixtures, the TaqPlus Precision PCR system tends to generate higher yields of PCR product than can be obtained using single-enzyme formulations.

With the introduction of Taq DNA polymerase in 1987,1 the polymerase chain reaction (PCR) amplification of DNA target regions has become an important and widely used tool of molecular biology. Since then, many additional thermophilic DNA polymerases and polymerase mixtures have been developed for use in PCR amplifications. Stratagene now offers four different thermophilic polymerases or polymerase mixtures for PCR: Taq DNA polymerase, Pfu DNA polymerase,** the TaqPlus Long PCR system,## (formerly TaqPlus DNA polymerase) and most recently, the TaqPlus Precision PCR system. All of these polymerases and polymerase systems are licensed for PCR, and each offers distinct characteristics and advantages.

Taq and Taq2000 DNA Polymerase for Reliability and Speed

Taq DNA polymerase has been the traditional workhorse enzyme of PCR amplification research. This enzyme was isolated originally from the thermophilic bacterium Thermus aquaticus strain YT1. Taq DNA polymerase exhibits both thermostable 5' - to 3'- polymerase and 5 - to 3 - exonuclease activities but lacks an associated 3 - to 5 - exonuclease-dependent proofreading activity. The relatively high error rate of Taq DNA polymerase has been attributed to the absence of an associated proofreading activity. Taq DNA polymerase exhibits a comparatively rapid rate of nucleotide incorporation, averaging approximately 2800 nucleotides per minute,2 which contributes to the reliability of this enzyme.

Stratagene provides both native Taq DNA polymerase, purified from T. Aquaticus YT1, as well as a fulllength recombinant version, Taq2000 DNA polymerase. Stratagene's highly purified Taq2000 DNA polymerase and associated PCR buffer have been shown to exhibit minimal artifactual smearing, which is commonly seen with competitors Taq DNA polymerase preparations using long (8 minute) extension times.

Pfu DNA Polymerase for Highest Fidelity and Thermostability

Pfu DNA polymerase is purified from the thermophilic archae Pyrococcus furiosus and exhibits both 5- to 3- polymerase and 3- to 5-exonucleasedependent proofreading activities. The proofreading activity associated with Pfu DNA polymerase contributes to its low PCR error rate. Several different methods, including forward mutation assays,4,5,6 DNA sequence analyses7 and denaturant gradient gel electrophoresis (DGGE) analyses8 have been used to show that Pfu DNA polymerase has the lowest error rate of any thermostable DNA polymerase studied to date. Pfu DNA polymerase also lacks terminal transferase activity and produces only blunt-ended PCR products, thereby making Pfu DNA polymerase the enzyme of choice for blunt-end PCR cloning with vectors such as the PCR-Script cloning vectors.9 In addition, Pfu DNA polymerase has unusually high thermostability, retaining more than 95% activity after 1 hour at 95C10 (half-life of approximately 12 hours at 95C) and can successfully amplify GC-rich templates requiring denaturing temperatures of 96-98C.11 A limitation of Pfu DNA polymerase is its relatively slow polymerization rate, incorporating approximately 550 nucleotides per minute.2 However, when used with a minimum extension time of 2 minutes per kb,12 this enzyme can successfully synthesize PCR products up to 12 kb in cloned Pfu reaction buffer13 and up to 25 kb in Taq reaction buffer (table 1).

PCR Product Length (kb)

Extension time required

Error Rates 1,2

Vector Target Yield3 Genomic Target Yield3 Min/kb target (x10-6)

(high)

(low)

(high)

(low) Pfu DNA polymerase

2+4

1.3

0-5

5-25

0-45

4-125 TaqPlus Precision system

1

3.9

0-10

10-15

0-5

5-10 TaqPlus Long system

0.5-1

7.6

0-25

25-35

0-12

12-18.5 Taq DNA polymerase

1+4

8.0

0-5

5-15

0-4

4-6

table 1

Stratagene provides both native and recombinant versions of Pfu DNA polymerase.14, 15 The high fidelity of Pfu DNA polymerase makes it ideal for amplifying products for applications such as cloning, sequencing, gene expression and sitedirected mutagenesis.

TaqPlus Long PCR System for Highest Yield and Long Targets

Certain mixtures of proofreading and nonproofreading DNA polymerases have been shown to significantly increase PCR product yield and enhance amplification of longer targets compared to single-enzyme formulations.16-18 The enhanced performance of polymerase mixtures is presumably due to the capacity of one enzyme to complement the inability of a second enzyme to extend a primer through potential template obstructions. These potential obstructions include mispaired bases that cause nonproofreading polymerases to stall prematurely and disassociate from the primer-template,16 abasic gaps that cannot be bridged by polymerases lacking terminal transferase activity19 and template secondary structures20 such as GC-rich hairpins.

The TaqPlus Long PCR system was developed for PCR amplifications of long DNA target regions (up to 35 kb) using relatively short extension times (0.5-1 minute per kb target). The system includes the TaqPlus Long DNA polymerase mixture, consisting of Taq2000 DNA polymerase and cloned Pfu DNA polymerases, and two reaction buffers. The low-salt buffer is recommended for relatively short target (010 kb) amplifications, while the high-salt buffer is optimal for longer target (535 kb) amplifications. The TaqPlus Long PCR system typically generates higher yields of both short and long PCR products than can be obtained using either component enzyme alone.21 Including Taq2000 DNA polymerase in the TaqPlus Long polymerase formulation ensures that artifactual background smearing is minimal when long extension times (8 minutes) are used.

figure 1

As with many commercially available two-polymerase mixtures, a potential limitation of the TaqPlus Long PCR system is its relatively low fidelity compared to Pfu DNA polymerase (figure 1). The TaqPlus Long PCR system exhibits an error rate similar to Taq DNA polymerase, the predominant component in the TaqPlus Long polymerase mixture. To address the needs of researchers requiring both high yields of PCR product and high replication fidelity, Stratagene has added the TaqPlus Precison PCR system to its expanding line of PCR products.

TaqPlus Long PCR System for Highest Yield and Long Targets

Certain mixtures of proofreading and nonproofreading DNA polymerases have been shown to significantly increase PCR product yield and enhance amplification of longer targets compared to single-enzyme formulations.16-18 The enhanced performance of polymerase mixtures is presumably due to the capacity of one enzyme to complement the inability of a second enzyme to extend a primer through potential template obstructions. These potential obstructions include mispaired bases that cause nonproofreading polymerases to stall prematurely and disassociate from the primer-template,16 abasic gaps that cannot be bridged by polymerases lacking terminal transferase activity19 and template secondary structures20 such as GC-rich hairpins.

The TaqPlus Long PCR system was developed for PCR amplifications of long DNA target regions (up to 35 kb) using relatively short extension times (0.5-1 minute per kb target). The system includes the TaqPlus Long DNA polymerase mixture, consisting of Taq2000 DNA polymerase and cloned Pfu DNA polymerases, and two reaction buffers. The low-salt buffer is recommended for relatively short target (010 kb) amplifications, while the high-salt buffer is optimal for longer target (535 kb) amplifications. The TaqPlus Long PCR system typically generates higher yields of both short and long PCR products than can be obtained using either component enzyme alone.21 Including Taq2000 DNA polymerase in the TaqPlus Long polymerase formulation ensures that artifactual background smearing is minimal when long extension times (8 minutes) are used.

As with many commercially available two-polymerase mixtures, a potential limitation of the TaqPlus Long PCR system is its relatively low fidelity compared to Pfu DNA polymerase (figure 1). The TaqPlus Long PCR system exhibits an error rate similar to Taq DNA polymerase, the predominant component in the TaqPlus Long polymerase mixture. To address the needs of researchers requiring both high yields of PCR product and high replication fidelity, Stratagene has added the TaqPlus Precison PCR system to its expanding line of PCR products.

Taqplus Precision PCR System for Highest Fidelity

The TaqPlus Precision PCR system consists of a unique mixture of Taq2000 DNA polymerase and cloned Pfu DNA polymerase and a PCR buffer, all optimized for highest PCR fidelity. Fidelity measurements of the TaqPlus Precision PCR system were carried out using a PCRbased forward mutation assay21,22 that utilizes the wellcharacterized lacIOZ target gene.23,24 figure 1 shows errorrate comparisons between Taq and Pfu DNA polymerases, the TaqPlus Long PCR system and the TaqPlus Precision PCR system. The TaqPlus Precision PCR system exhibits a mean error rate of 3.9 x 106 mutations per bp per duplication, which is 3-fold higher than the error rate of Pfu DNA polymerase, but 1.9-fold and 2.1-fold lower than the error rates of the TaqPlus Long PCR system and Taq DNA polymerase, respectively. The TaqPlus Precision reaction buffer, provided in this system is also critical for optimal PCR fidelity (data not shown). Moreover, the TaqPlus Precision PCR system was found to exhibit the highest average replication accuracy that has been achieved using commercially available DNA polymerase mixtures.25

Figure 2

The TaqPlus Precision PCR system also tends to generate higher yields of PCR product than can be obtained with either Taq DNA polymerase or Pfu DNA polymerase. Figure 2 shows a typical comparison of PCR product yields for Taq DNA polymerase and the TaqPlus Precision PCR system for various target systems. The TaqPlus Precision PCR system successfully synthesizes vector PCR targets up to 15 kb in length and genomic PCR targets up to 10 kb in length using a relatively short extension time of 1 minute per kb of DNA target.

Comparison of PCR Performance

Table 1 summarizes the recommended PCR extension time, error rate, relative PCR product yield and useful target length for Stratagene's four thermophilic DNA polymerases and polymerase mixtures. Taq DNA polymerase is relatively fast and reliable and is suitable for amplifying targets 4 kb and smaller when fidelity is not a concern. Pfu DNA polymerase has unusually high thermostability and the lowest error rate of any thermostable DNA polymerase studied to date. Clearly, Pfu DNA polymerase is the enzyme of choice for PCR applications requiring high fidelity or amplification through GCrich regions, when relatively long extension times can be used (2 minutes per kb of DNA target). Although Pfu DNA polymerase can amplify up to 25 kb, the yield of PCR product is generally lower than can be obtained with polymerase mixtures. The TaqPlus Long PCR system is ideally suited for generating high yields of PCR product and amplifying long targets up to 35 kb when fidelity is not the primary consideration. Finally, the TaqPlus Precision PCR system exhibits an error rate significantly lower than Taq DNA polymerase, the TaqPlus Long PCR system and other commercially available sbased polymerase mixtures.25 The TaqPlus Precision system is recommended for relatively rapid, highfidelity amplification of PCR targets up to 15 kb when Pfu DNA polymerase requires too long an extension time or yields are insufficient.

Conclusions

Cycling time, fidelity, target length and product yield are all important considerations when selecting a DNA polymerase or twopolymerase mixtures for PCR. Stratagene offers four different thermostable DNA polymerases and polymerase mixtures that are uniquely suited for particular PCR applications. The TaqPlus Precision PCR system, the latest addition to Stratagene's line of PCR products, offers researchers improved PCR fidelity.

REFERENCES

  1. Mullis, K.B., and Faloona, F.A. (1987) Methods Enzymol. 155: 335350.

  2. Hogrefe, H. Unpublished.

  3. Nielson, K.B., et al. (1996) Strategies 9: 78.

  4. Lundberg, K.S., et al. (1991) Gene 108: 16.

  5. Flaman, J.M., et al. (1994) Nucleic Acids Res. 22: 32593260.

  6. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996) Nucleic Acids Res. 24: 35463551.

  7. Marykwas, D.L., and Passmore, S.E. (1995) Proc. Natl. Acad. Sci. USA 92: 1170111705.

  8. Cha, R.S., and Thilly, W.G. (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

  9. Sanchez, T., Zheng, C.F., and Bauer, J.C. (1996) Strategies 9: 4446.

  10. Bergseid, M., et al.(1991) Strategies 4: 3435.

  11. Chong, S.S., Eichler, E.E., Nelson, D.L., and Hughes, M.R. (1994) Amer. J. Med. Genet. 51: 522526.

  12. Nielson, K.B., Costa, G.L., and Braman, J. (1996) Strategies 9: 2425.

  13. Nielson, K., Braman, J., And Kretz, K. (1995) Strategies 8: 26.

  14. Cline, J., Nielson, K.B., Scott, B., And Mathur, E. (1992) Strategies 5: 50.

  15. Scott, B., Nielson, K.B, Cline, J., And Kretz, K. (1994) Strategies 7: 6263.

  16. Barnes, W. (1994) Proc. Natl. Acad. Sci. USA 91: 22162220.

  17. Cohen, J. (1994) Science 263: 15641565.

  18. Cheng, S., et al.(1994) Proc. Natl. Acad. Sci. USA 91: 56955699.

  19. Hu,G. (1993) DNA Cell Biol. 12: 763770.

  20. Eckert, K.A., and Kunkel, T.A. (1993) J. Biol. Chem. 268: 13462.

  21. Nielson, K., Scott, B., Bauer, J.C., and Kretz, K. (1994) Strategies 7: 64.

  22. Cline, J. Braman, J. And Kretz, K. (1995) Strategies 8:2425.

  23. Kohler, S.W., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 79587962.

  24. Provost, G.S., et al. (1993) Mutat. Res. 288: 133.

  25. Guide to Pfu DNA Polymerase, Stratagene, 1996.


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