QIAGEN Multiplex PCR Plus Kit

• Successful multiplex PCR for advanced applications
• Consistent and reliable results
• Rapid establishment of multiplex PCR assays without optimization
• Faster reaction times and greater convenience

The QIAGEN Multiplex PCR Plus Kit is based on QIAGEN's proprietary multiplex PCR technology and ensures PCR success at the first attempt. The preoptimized master mix includes HotStarTaq Plus DNA Polymerase and an innovative PCR buffer system, specially developed for multiplex PCR. The stringent hot-start mechanism provided by HotStarTaq Plus DNA Polymerase and the unique composition of the buffer increases multiplex reaction specificity by preventing extension of nonspecifically annealed primers and primer dimers.

High specificity and sensitivity, without the need for optimization

The QIAGEN Multiplex PCR Plus Kit outperformed kits tested from other suppliers and delivers high specificity and sensitivity in multiplex PCR applications, eliminating the need for optimization of PCR parameters (see figures " Efficient 19-plex PCR" and " Successful 16-plex PCR over a wide range of template amounts"). Suboptimal PCR can be improved with Q-Solution — an additive for the amplification of GC-rich templates — also provided with the kit.

Reliable results lead to time and cost savings for advanced applications

The QIAGEN Multiplex PCR Plus Kit can be used successfully for a wide variety of advanced applications (see table). The simple reaction setup, fast procedure, and ease of use provided by the kit ensure reproducible results faster, without the need for optimization of PCR parameters. Multiplex assay development is straightforward and easy, ensuring significant time and cost savings in routine research.

HotStarTaq Plus DNA Polymerase specifications

The QIAGEN Multiplex PCR Plus Kit is based on QIAGEN's proprietary multiplex PCR technology and is provided in an easy-to-use master mix format. All of the kit components are specially developed to ensure maximum ease of use and speed, delivering consistently reliable results (see table). The Multiplex PCR Master Mix contains preoptimized concentrations of HotStarTaqPlus DNA Polymerase and MgCl₂, as well as dNTPs and an innovative PCR buffer.

Critical factors for successful multiplex PCR —  consistent and reliable results for various applications

Multiplex PCR saves time and reagents for researchers performing large numbers of PCR reactions and is widely used in genotyping and DNA or cDNA testing applications in research, forensic, and molecular testing laboratories. Applications include typing and analysis of transgenic organisms and pathogens, amplification and analysis of microsatellites, and detection of regions for SNPs or mutations, as well as metagenomics studies. However, multiplex PCR is a highly demanding technique. Optimization of PCR is the main factor influencing the success or failure of multiplex PCR. The varying hybridization kinetics of different primer pairs in multiplex PCR can lead to problems such as amplification bias. Primers that bind with high efficiency utilize more of the amplification reaction components, thereby reducing the yield of other PCR products. Due to the larger number of primers, there is also a greater risk of primer dimer formation and nonspecific priming. Not addressing these challenges leads to poor sensitivity, nonspecific amplification, and biased amplification of selected targets — and therefore inconsistent and unsatisfactory results. Overcoming these bottlenecks requires tedious and time-consuming optimization steps, resulting in increased costs. The QIAGEN Multiplex PCR Plus Kit eliminates these challenges and easily works at the first attempt. The unique composition of the Multiplex PCR Master Mix ensures highly specific and sensitive amplification, even of difficult-to-amplify targets. The kit enables rapid and successful establishment of all multiplex PCR assays, significantly saving time and costs (see figure " Successful multiplex PCR without the need for optimization").

HotStarTaq Plus DNA Polymerase

HotStarTaq Plus DNA Polymerase is a modified form of Taq DNA polymerase and has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle. Optimization of reaction conditions is not required. Multiplex assays can also be easily set up at room temperature. HotStarTaq DNA Polymerase is activated by a 5-minute incubation at 95°C which can be incorporated into any existing thermal-cycler program.

Multiplex PCR Buffer

This special buffer contains an optimized combination of K⁺ and NH₄⁺, as well as the unique PCR additive, Factor MP, which increases the local concentration of primers at the template (see figure " Stable and efficient annealing"). Nonspecific annealing is minimized and parallel amplification of all targets is successful — even with very low template amounts (see figure " Successful 16-plex PCR over a wide range of template amounts"). Together with K⁺ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase. The innovative buffer maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle. Owing to a uniquely balanced combination of KCl and (NH₄)₂SO₄, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg²⁺ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg²⁺ concentration is therefore often minimal or not required.

The kit also includes Q-Solution, a unique additive that promotes amplification of difficult-to-amplify targets such as GC-rich regions or templates with a complex secondary structure. Highly versatile CoralLoad Dye — also provided with the kit — further increases handling convenience by improving pipetting visibility and subsequent gel loading and visualization of DNA migration (see figure " Easy PCR setup and convenient DNA visualization").

In contrast to alternative approaches — which fail even after lengthy optimization — the QIAGEN Multiplex PCR Plus Kit enables success in multiplex PCR at the first attempt using a single protocol (see figure " Successful multiplex PCR without the need for optimization").

Straightforward and fast downstream analysis

Downstream analysis of multiplex PCR products obtained with the QIAGEN Multiplex PCR Plus Kit is straightforward and fast using a variety of methods. Whether analysis is performed using agarose gels, capillary sequencers, or using the the QIAxcel Advanced System, all amplicons can be easily visualized and individual fragments can be reliably distinguished (see figure " Efficient 19-plex PCR").

Convenient kit format

The QIAGEN Multiplex PCR Plus Kit provides a ready-to-use, preoptimized master mix for greater convenience. Use of a master mix saves time, simplifies handling for reaction setup, and increases reproducibility by eliminating many possible sources of pipetting errors and contamination — pipetting steps are minimized and tedious calculations are eliminated. Only primers and template need to be added to prepare the final amplification mix. The master mix can be stored at 2–8°C, allowing even faster setup of multiplex PCR assays. Reactions can be set up at room temperature, ensuring greater convenience and ease of use. HotStarTaqPlus DNA Polymerase is activated by a 5-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. CoralLoad Dye supplied with the kit ensures greater convenience by improving pipetting visibility during PCR setup and visualization of DNA migration on a gel.

Supplementary protocol for preamplification of up to 80 targets