For ultrafast, hot-start mediated, long-range PCR

Features

Exceptionally fast long-range PCR significantly streamlines workflows
A polymerase-proofreader blend with 10x higher fidelity than Taq ensures clear sequencing results
Moderate multiplexing of up to 6 targets facilitates simultaneous analysis of various regions
Superior specificity and sensitivity – antibody-mediated hot-start of polymerase and exonuclease
Visual pipetting control and gel tracking dyes monitor successful procedure
Automation-ready, stable, room-temperature reaction setup

Product Details

The UltraRun LongRange PCR Kit provides a convenient format for highly sensitive and specific long-range PCR for up to 30 kb using any DNA or cDNA template. The ready-to-use master mix contains a blend of hot-start controlled Taq polymerase, a proofreader and other additives, which provide exceptional speed over current long-range PCR kits and moderate multiplexing capability. The kit is, therefore, an ideal choice for long amplicon sequencing in immunology, cancer and microbial workflows. Additional features include visual pipetting control, room-temperature stability during and after reaction setup and a 4x-concentrated master mix format for higher sample input volumes. Furthermore, the optional Q-Solution allows amplification of GC-rich and other challenging targets.

Performance

A sophisticated buffer composition and enzyme blend provide long-range amplification of up to 30 kb at an exceptional speed using a combined 30 seconds/kb annealing-extension step. High yields can be obtained from as little as a few picograms of template. This greatly improves screening workflows, easily enabling long-range PCR with subsequent analysis in a day. The robust formulation allows automated reaction setup at room temperature because both polymerase and proofreader activities are controlled by antibody-mediated hot-start, preventing any damage to template, primers or products arising from premature enzyme activity. The uracil-literate proofreader shows high template affinity providing long reads and 10x higher fidelity than Taq. The optional use of Q-Solution enables amplification of difficult or GC-rich targets, which is particularly beneficial in long-range PCR. Furthermore, the master mix formulation allows moderate multiplexing of up to 6 targets in a range of approximately 1–9 kb. The 4x master mix concentration along with integrated visual pipetting and gel loading dyes further improve efficiency in genotyping and genetic testing workflows.

Principle

The UltraRun LongRange PCR Kit provides a convenient 4x master mix formulation, an orange Master UCP Mix Tracer, a blue UCP Template Tracer and Q-Solution (for optional use) in separate vials.

The ready-to-use master mix consists of a blend of enzymes – Taq DNA polymerase and a polymerase with proofreading capability – which improves hands-on time and process robustness, a sophisticated PCR buffer optimized for long PCR and dNTP mix. At low temperatures, both polymerase and proofreader activities are kept in an inactive state by antibodies. This provides a stringent hot-start and prevents any enzymatic activity until heat activation at 93°C, thereby avoiding primer or DNA damage from untimely proofreader activity. Due to this functionality, complete reactions, including primers and template, are stable at room temperature for longer periods for convenient process handling. Furthermore, the 4x master mix concentration allows greater flexibility for template input volumes over common 2x master mixes and the dyes in the master mix formulation enable direct loading of PCR reactions onto agarose gels for analysis.

The UltraRun LongRange PCR Kit includes Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is non-toxic and PCR purity is guaranteed.

The blue and orange dyes in the UCP Template Tracer and in the UCP Master Mix Tracer, respectively, allow visual tracking of pipetted samples during the PCR setup, to prevent errors. When the blue template is added to the orange PCR Master Mix, the color changes to green, confirming that the sample was added (see figure  Visual pipetting control). The use of these tracers is optional. Reactions can be directly loaded onto agarose gels after cycling. Each tracer dye allows monitoring of the loading process and efficient tracking of the subsequent electrophoresis.

Procedure

A high-template input volume can be used with the UltraRun LongRange PCR Kit due to its 4x-concentrated master mix formulation, further boosting sensitivity.

The orange Master Mix Tracer is optionally added to the master mix or PCR buffer. When template dyed with the blue Template Tracer is added to the master mix, the color of the solution changes from orange to green, providing a visual indication of correct pipetting and reaction setup (see figure  PCR procedure).  The tracer dyes conveniently allow direct loading on agarose gels after PCR and efficient tracking of electrophoresis. The dyes run at approximately 50 bp (orange) and 4000 bp (blue) on a 1% agarose gel (see figure  Easy visualization).

The versatile buffer system allows ultrafast PCR using a combined 30 seconds/kb annealing-extension step. It also eliminates the need for optimization or changing buffer components. The Q-Solution can be optionally used to alter the melting behavior and enable amplification of challenging templates. The reaction can be fully set up at room temperature without ice and using automated pipetting, thanks to the hot-start mechanism.

To further streamline PCR workflows, we recommend the QIAgility instrument for automated reaction set up. For microbiome analysis, QIAGEN offers a range of dedicated products including the DNeasy Power Soil Pro Kit for sample extraction, QIAseq 16S/ITS Region Panels for next-generation sequencing and QIAGEN CLC Genomics Pro Suite software for bioinformatics analysis.

Applications

The UltraRun LongRange PCR Kit is ideally suited for procedures involving long target amplification, including long amplicon sequencing in immunology, cancer and microbial workflows. Analysis of oncogenes, HLA or pan-bacterial and fungal sequences like 16S, 23S and ITS can be substantially streamlined using the high yields from ultrafast and automatable long-range PCR.