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").
Advantages of the QIAGEN Multiplex PCR Plus Kit
|HotStarTaq Plus DNA Polymerase ||Highly specific and sensitive amplification |
5-minute activation time
|Multiplex PCR Plus Buffer with Factor MP ||Amplification of all targets in parallel |
No optimization of PCR parameters needed
One single protocol for all multiplex assays
|CoralLoad Dye ||Easy visualization during pipetting |
Immediate gel loading
Visualization of DNA migration
|Q-Solution ||Amplification of difficult targets |