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Tech Note: Superior amplification of GC-rich templates with PrimeSTARâ„¢ GXL DNA Polymerase

TECH NOTE

Superior amplification of GC-rich templates with PrimeSTAR GXL DNA Polymerase

Data kindly provided by: Alexander Ebralidze, Scientist (Beth Israel Deaconess Medical Center)

Overview

PCR amplification of GC-rich templates is often hindered by formation of secondary structures and the requirement for high melting temperatures. PrimeSTAR GXL DNA Polymerase is a high fidelity PCR enzyme that allows efficient amplification of the most challenging templates. With this enzyme, amplification of GC-rich templates is possible without reaction optimization or inclusion of additives such as DMSO or Betaine.

In this experiment, PrimeSTAR GXL DNA polymerase was compared to other commercially available enzymes for the amplification of a target with high GC content. Only the PrimeSTAR GXL enzyme was able to efficiently amplify the GC-rich template without additives or special reaction conditions.

Results

Under standard reaction conditions, only PrimeSTAR GXL DNA polymerase produced a single amplification product of the expected size (Figure 1). PCR product was successfully obtained with the competitor enzymes only when the reactions included DMSO (5% final) and Betaine (1 M final) (Figure 2, left). Very little product was obtained with PrimeSTAR GXL polymerase in the presence of these additives (Figure 2, right).

 

Figure 1. PCR products from the standard 3 step PCR reaction were analyzed in a 1.2% agarose gel. Figure 2. PCR products from the rapid 3-step PCR reaction were analyzed in a 1.2% agarose gel.
Figure 1. Amplification products obtained under standard reaction conditions. The expected product size is 603 bp. Pfu: Pfu polymerase; Co. A: Company A master mix; Co. D: Company D master mix; PS: PrimeSTAR GXL polymerase. Figure 2. Amplification products obtained from reactions containing DMSO and Betaine. The expected product size is 603 bp. Pfu: Pfu polymerase; Co. A: Company A master mix; Co. D: Company D master mix; PS: PrimeSTAR GXL polymerase.

Conclusions

In this experiment, PrimeSTAR GXL DNA polymerase was the only polymerase that was able to amplify a sequence from template DNA with a GC content >75% without additives. PrimeSTAR was able to amplify this target without special reaction conditions (a standard 2-step PCR protocol was used) and without additives. In contrast, all of the other competitor enzymes tested were only able to amplify when DMSO and Betaine were added to the reaction. These results indicate that PrimeSTAR GXL polymerase is ideal for optimization-free PCR amplification of GC-rich targets.

Materials and Methods

A 603 bp region of the CEBPA gene promoter that contains 75.29% GC was amplified from human genomic DNA isolated from K562 cell line using either PrimeSTAR GXL DNA Polymerase, a master mix from Company A, a master mix from Company D, or Pfu DNA Polymerase. Reactions were assembled according to the manufacturer’s recommendations. All reactions were run on a MultiGene Thermal Cycler (Labnet) following the cycling conditions in Table 1. Amplified products were analyzed by gel electrophoresis.

Table 1. PCR cycling conditions.

Learn more about PrimeSTAR GXL DNA Polymerase >>

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