The Type-it Mutation Detect PCR Kit is based on proprietary QIAGEN Multiplex Technology and is provided in a ready-to-use master mix format. The master mix contains optimized concentrations of HotStarTaq Plus DNA Polymerase, MgCl2, and dNTPs, and an innovative PCR buffer, specially developed for multiplex PCR-based detection of mutations or for preamplification of genomic SNP loci. It also includes the novel additive Factor MP, as well as a balanced combination of salts and additives. Together, these components enable comparable efficiencies for annealing and extension of all primers in the reaction (see figure " Stable and efficient annealing"), providing reliable high-yield multiplex amplification of all fragments in parallel. The kit includes dedicated protocols for the detection of mutations, as well as step-by-step advice on template amounts, cycle numbers, and PCR conditions and instrument details for different downstream analysis platforms such as agarose gels, capillary sequencers, the Agilent Bioanalyzer, and the QIAxcel Advanced System.
The combination of all components provided in the master mix and the dedicated protocol result in highly specific amplification of all fragments in parallel. Subsequent analysis is straightforward and easy and can be carried out on agarose gels, automated electrophoresis instruments, and also by high-resolution capillary sequencing.
Type-it Mutation Detect PCR Buffer
The Type-it Mutation Detect PCR Buffer ensures comparable amplification efficiency for all amplicons in a high-plex grade multiplex experiment and allows more mutation targets to be combined without the loss of amplification efficiency for any of the targets. In contrast to conventional PCR reagents, the Type-it Mutation Detect PCR Buffer contains a specially developed, balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer-annealing conditions over a wider range of temperatures and Mg2+ concentrations than conventional PCR buffers. The need to optimize PCR by varying the annealing temperature or the Mg2+ concentration is dramatically reduced, or often not required. Commonly employed optimization procedures for multiplex PCR are virtually eliminated. The buffer also contains the synthetic Factor MP (see figure " Stable and efficient annealing"), which allows efficient primer annealing and extension, irrespective of primer sequence. Factor MP increases the local concentration of primers at the DNA template and stabilizes specifically bound primers.
The Type-it Mutation Detect PCR Kit is supplied with Q-Solution. This innovative PCR additive facilitates amplification of difficult templates by modifying the melting behavior of DNA. Use of this unique reagent will often enable or improve suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer-template system and is not toxic.
The Type-it Mutation Detect PCR Kit is supplied with CoralLoad Dye (see figure " CoralLoad Dye"), which contains a gel-loading reagent and two gel-tracking dyes that improve pipetting visibility and facilitate estimation of DNA migration distance, as well as optimization of agarose gel run time. When using CoralLoad Dye, in the multiplex PCR reaction, the amplicons can be directly loaded onto an agarose gel or the QIAxcel Advanced System without prior addition of loading buffer. CoralLoad dyes do not interfere with most downstream enzymatic applications. However, for reproducible results, purification of PCR products prior to enzymatic manipulation is recommended.
If using capillary sequencers for detection, CoralLoad Dye must not be used.