Long Range PCR

Long range PCR typically refers to the amplification of DNA fragments in excess of 5 kb, using a blend of thermostable DNA polymerases that allows for the amplification of longer fragments than those that can be achieved with a single enzyme. Enzyme blends consist primarily of an A-family DNA polymerase (e.g. Taq), combined with a small amount of a B-family or proofreading DNA polymerase (e.g. Pfu). Both enzymes possess 5’ – 3’ DNA-dependent DNA polymerase activity, but only the B-family polymerase possesses 3’ – 5’ exonuclease or proofreading activity.

A-family DNA polymerases like Taq are unable to efficiently extend beyond misincorporated bases due to their lack of proofreading activity. Mismatched base pairing therefore leads to truncated products that accumulate during PCR, reduce the effective template copy number and contribute to reaction failure if the target is long and/or the concentration of template DNA is low. Wild-type B-family DNA polymerases are extremely accurate due to their ability to repair mismatches but do not perform well over longer target distances or with low template concentrations because their 3’ – 5’ exonuclease (proofreading) activity destroys primers and affects sensitivity. By combining the robustness of A-family polymerases with misincorporation repair through the 3’ - ‘5’ exonuclease activity of the minor (B-family polymerase) component, long-range blends are capable of long and sensitive PCR. The error rates of enzyme blends are typically two- to six-fold lower than that of Taq, but are significantly higher than that of pure B-family DNA polymerases.

Solix Long Range DNA Polymerase is a blend of KAPA Taq DNA polymerase and the novel, engineered Solix HiFi B-family DNA polymerase. In the HotStart formulation, Solix Taq is combined with a proprietary antibody that blocks Taq activity until the initial denaturation step. This prevents the formation of spurious reaction products during reaction setup, and increases overall reaction efficiency.

Solix Long Range and Long Range HotStart PCR Kits offer:

  • Efficient amplification of long DNA fragments (up to 25 kb).
  • Improved sensitivity in the amplification of both short and long amplicons.
  • Two- to six-fold higher in fidelity than wild-type Taq.
  • The robustness of A-family polymerases, i.e. typically less optimization of reaction parameters than required for B-family polymerases.
  • dA-tailed reaction products that can be cloned into TA-cloning vectors.

Advanced Applications of Long Range PCR in Cutting-Edge Research :

Long Range PCR is not just limited to amplification but is a cornerstone for innovative research in various fields, offering an indispensable tool for high-accuracy studies requiring long DNA fragments. This powerful technique is instrumental in unraveling complex genetic processes, sequencing large regions of the genome, and investigating difficult-to-amplify DNA regions.

Why Researchers Choose Long Range PCR ?

  • High Sensitivity for Low DNA Input: Long Range PCR is especially beneficial for researchers dealing with low DNA inputs or precious samples, ensuring high sensitivity even when template DNA is scarce.
  • Minimized Primer-Dimer Formation: A common challenge in long-template PCR is the formation of primer-dimers, which often complicate amplification. The Solix Long Range Polymerase effectively minimizes primer-dimer formation, ensuring that amplification is focused on the target DNA.
  • Reproducibility and Scalability: Whether working on small-scale projects or large genomic surveys, Long Range PCR offers the reproducibility and scalability needed to manage diverse genetic research tasks. The ability to amplify large DNA fragments across multiple conditions enhances the depth of data analysis and results interpretation.
  • Support for Complex Genomic Studies: With the flexibility to amplify long and complex genomic sequences, Long Range PCR is crucial in genomic mapping, identifying large-scale mutations, and examining gene regulatory regions that traditional PCR cannot easily address.

Versatile Use in Clinical and Diagnostic Research

In clinical and diagnostic research, Long Range PCR offers new possibilities for detecting genetic mutations, examining gene duplications, or amplifying genomic regions that might be difficult to target with standard PCR techniques.

  • Cancer Research: Long Range PCR plays an essential role in the identification of cancer-related mutations or the detection of large gene rearrangements in oncology research.
  • Genetic Disorder Investigations: Researchers use Long Range PCR to analyze genes linked to inherited genetic disorders, enabling the identification of large deletions or mutations that standard PCR may miss.

Key Benefits of Long Range PCR :

  • Efficient Amplification of Large Templates: Whether studying large genes or long regions of the genome, Long Range PCR is engineered to perform with high efficiency, even in the face of difficult DNA structures or low template DNA.
  • Accurate Detection in Complex Genomic Regions: The Solix Long Range Polymerase enables precision in regions with high GC content or secondary structures that hinder traditional PCR techniques.
  • Seamless Integration with Other Techniques: Compatible with various next-generation sequencing (NGS) methods, Long Range PCR can be used to prepare DNA templates for deeper genomic analysis.