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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

Introduction Results Conclusions Methods

Introduction  

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 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. PCR products from the rapid 3-step PCR reaction were analyzed in a 1.2% agarose gel.

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 PCR enzyme able to amplify a sequence from template DNA with a GC content >75% without additives. PrimeSTAR GXL DNA polymerase was able to amplify this target without special reaction conditions (a standard two-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 DNA Polymerase is ideal for optimization-free PCR amplification of GC-rich targets.

Methods  

A 603-bp region of the CEBPA gene promoter that contains 75.29% GC content was amplified from human genomic DNA (isolated from the 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 below. Amplified products were analyzed by gel electrophoresis.

Table 1. PCR cycling conditions
PrimeSTAR GXL polymerase (2-step PCR) Company A, Company D, and Pfu polymerase (3-step PCR)
98°C 10 sec 35 cycles 95°C 30 sec 35 cycles
60°C 15 sec
68°C 20 sec
72°C 30 sec

Related Products

Cat. # Product Size License Quantity Details
R050B PrimeSTAR® GXL DNA Polymerase 1,000 Units USD $749.00

A hot-start, high-fidelity PCR enzyme, PrimeSTAR GXL DNA Polymerase excels in reactions with GC-rich templates, excess template, and long amplicons up to 30 kb (GXL). The polymerase is supplied with separate tubes of optimized buffer (Mg2+ plus), and dNTPs. Cat. # R050B contains 4 of Cat. # R050A. Please refer to Cat. # R050A for complete product documentation and resources.

Also available as a premix.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Examples of product yield on GC-rich templates: comparison of PrimeSTAR GXL DNA Polymerase with four other commercially available high-fidelity DNA polymerases

Examples of product yield on GC-rich templates: comparison of PrimeSTAR GXL DNA Polymerase with four other commercially available high-fidelity DNA polymerases
Examples of product yield on GC-rich templates: comparison of PrimeSTAR GXL DNA Polymerase with four other commercially available high-fidelity DNA polymerases. Company T’s enzyme includes buffers optimized for GC-rich templates.

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PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA)

PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA)
PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA).

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PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases

PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases
PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases.


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