Accuracy and Efficiency for Any PCR Cloning Project

Successful PCR cloning demands accurate amplification. Any errors due to misincorporation of bases during PCR can adversely affect expression levels. Accuracy, however, should not be the only concern; cloning efficiency is also an important consideration. High cloning efficiency results in greater incorporation of the PCR product into the vector of choice. Taq-amplified products contain a 3’-A at the end of PCR products, while products amplified with proofreading enzymes do not. Many commercially available PCR enzymes labeled “high fidelity” are actually blends of Taq and a proofreading enzyme. These enzyme blends produce a mixture of PCR products, some with 3’-A overhangs and some with blunt ends. Such product mixtures exhibit inferior cloning efficiencies compared to PCR products amplified with a single polymerase. Table 1 describes the ends of PCR products amplified with various commercially available PCR enzymes.

Stratagene’s portfolio of high-performance PCR enzymes includes two polymerases formulated for high level accuracy and cloning efficiency. PfuUltra™ hotstart DNA polymerase* exhibits the lowest error rate of any commercially available PCR enzyme, and produces PCR products with blunt ends. Easy-A™ high-fidelity PCR cloning enzyme** provides a high level of accuracy and produces products with 3’-A overhangs for TA/UA cloning. Figure 1 illustrates the ease of cloning of PCR products amplified with these enzymes. Both polymerases are available in a 2X master mix format for maximum convenience and throughput.

The Highest Level of Accuracy Available

With an error rate of 4.3 x 10^-6, PfuUltra hotstart DNA polymerase is the most accurate PCR enzyme available1 (see accuracy in Table 1). This powerful enzyme is formulated with Stratagene’s exclusive ArchaeMaxx™ polymerase-enhancing factor*** providing higher yields and greater target-length capability. Products amplified with PfuUltra DNA polymerase contain blunt ends, providing high cloning efficiencies into any blunt-ended vector. No additional polishing steps are necessary to prepare amplified products for the cloning step.

Figure 1
Cloning of PCR Products Amplified with Easy-A™ PCR Cloning Enzyme or PfuUltra™ DNA Polymerase

REFERENCE

1. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996) Nucleic Acids Res. 24: 3546-3551.

*U.S. Patent Nos. 6,489,150, 6,444,428, 6,379,553, 6,333,165, 6,183,997, 5,948,663, 5,866,395, 5,556,772, 5,545,552 and patents pending

See license reference 9

** Patents pending

*** U.S. Patent Nos. 6,444,428, 6,379,553, 6,333,165, 6,183,997, and patents pending

Efficient, Accurate TA/UA Cloning

The Easy-A high-fidelity PCR cloning enzyme provides a high level of accuracy, second only to PfuUltra DNA polymerase. Unlike other high-fidelity enzymes, however, Easy-A PCR cloning enzyme adds a 3’-A overhang to the end of each product with the same efficiency as Taq. The result is amplified products with the TA/UA cloning efficiency of Taq and the low error rate of Pfu DNA polymerase. No additional steps, such as A-addition kits or incubation with Taq, are necessary prior to cloning. The Easy-A PCR cloning enzyme is also available in our new RT-PCR kit for simplified RT-PCR cloning. See article on page 26.

Don’t Sacrifice Accuracy for Efficiency

Taq DNA polymerase can provide high cloning efficiency, but exhibits a relatively high error rate. Some commercially available “high fidelity” enzymes provide increased accuracy over Taq, but exhibit lower cloning efficiencies. High-performance PCR enzymes from Stratagene can provide both accuracy and efficiency in one step, for superior results in any PCR cloning project.