Introduction
The QIAGEN LongRange PCR Kit combines a powerful polymerase blend with an innovative buffer system designed for efficient amplification of long targets. The LongRange PCR Enzyme Mix is a blend of thermostable DNA polymerases with enhanced processivity. This provides a very high extension rate in addition to proofreading ability, leading to increased fidelity. The LongRange PCR Buffer is designed to enable long-range PCR without the need for optimization, allowing successful amplification of genomic targets longer than 20 kb and episomal targets up to 40 kb in size. It also ensures reliable amplification of smaller fragments of 0.1–10 kb, using a simplified, faster, optimized protocol. The resulting amplicons can be easily detected and analyzed on QIAGEN’s multicapillary electrophoresis system — the QIAxcel.
Unique buffer formulation for outstanding long-range PCR success
Despite the fact that PCR amplification is performed routinely and that thousands of PCR protocols have been developed, researchers still encounter technical difficulties with PCR experiments and often fail to obtain specific amplification products, particularly if the amplicon exceeds a certain length. PCR products of up to 4 kb can be routinely amplified using standard PCR protocols. However, without lengthy optimization procedures, amplification of PCR products longer than 4 kb often fails. Reasons for failure include nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions — all factors which have a greater effect on the amplification of longer PCR products than on shorter ones.
Preventing DNA damage, such as DNA depurination, is of particular importance for amplification of long PCR products, as a single DNA lesion within the template is sufficient to stall the PCR enzyme. DNA damage during PCR cycling can be minimized with specific buffering substances that stabilize the pH of the reaction. The LongRange PCR Buffer provided with the kit is designed to ensure long-range PCR — without the need for optimization — enabling reliable amplification of genomic targets longer than 20 kb and episomal targets up to 40 kb in size. Its unique zwitterionic formulation improves pH buffering at high temperatures (72–94°C), thus minimizing pH-driven template degradation. DNA preincubated in the LongRange PCR Buffer shows similar PCR product yields compared to undamaged control DNA, demonstrating that the protective function of the buffer system provides an optimal reaction environment for the amplification of long PCR products.
Compared to long-range buffers from other suppliers, the advantages of QIAGEN’s LongRange PCR Buffer are clearly visible. As a result of its protective function, higher yields of amplified products are obtained and overall longer products can be amplified (see figures
High yields of very long PCR products and
LongRange PCR Buffer protects genomic DNA from excessive damage).
Effortless analysis of DNA fragments up to 10 kb in size
Currently, the most commonly used method for nucleic acid separation is gel electrophoresis, using manually poured slab gels. However, this method is highly labor-intensive and exposes users to hazardous chemicals such as ethidium bromide. Analysis of long-range PCR products of up to 10 kb length is made easier using the QIAxcel Advanced System which fully automates high-resolution capillary electrophoresis of up to 96 samples without manual intervention (see figures
QIAxcel Advanced System). It also ensures greater safety as exposure to hazardous chemicals such as ethidium bromide is minimized. Standardization is achieved through the use of pre-cast gel cartridges that allow up to 96 samples to be run using exact electrophoresis conditions. Therefore, the QIAxcel Advanced speeds up nucleic acid analysis applications by eliminating slab-gel analysis while streamlining the entire sample purification and analysis workflow.
Summary
The QIAGEN LongRange PCR Kit includes all of the components required for the reliable and high-yield amplification of long PCR fragments of up to 40 kb in size (see table). The novel buffer system ensures high specificity and high yield due to a novel zwitterionic formulation which exhibits a protective function against DNA depurination during the amplification process. Resulting amplicons of up to 10 kb length can be easily detected and analyzed using the novel QIAxcel System, making the entire procedure easy, fast, and straightforward, which is particularly significant in genotyping studies involving detection of gross alterations of the genome.
For sensitive and accurate long-range two-step RT-PCR, discover the QIAGEN LongRange 2Step RT-PCR Kit which includes all components necessary for long-range cDNA preparation.
For detailed information about typical PCR challenges and QIAGEN’s optimized solutions, download our comprehensive brochure. For further information about the QIAxcel, download the brochure. Learn more about our diverse PCR portfolio.
Unique features of the QIAGEN LongRange PCR Kit
| Applications |
Cloning, genotyping, sequencing, standard PCR of up to 40 kb fragments |
| Enzyme components |
DNA polymerase, high-fidelity enzyme |
| Minimal optimization required |
Unique buffer system and optimized protocols |
| Depurination protection of templates |
Unique buffer system with a zwitterionic formulation |
| Amplification of GC-rich templates |
Q-Solution provided |
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.