Type-it Mutation Detect PCR Kit

For accurate and reliable multiplex PCR analysis of mutations

Features

  • Successful and reproducible analysis of multiple mutations
  • Multiplex PCR assay development without optimization
  • Specific and sensitive coamplification of all fragments
  • Optimized protocol for fast and reliable results
  • Easy instructions for use with various downstream analysis platforms
Type-it Mutation Detect PCR Kit (200)

Cat. No. / ID: 206343

For 200 x 25 μl reactions: Type-it Multiplex PCR Master Mix, 5x Q-Solution, RNAse-Free Water, and 10x CoralLoad Dye
€214.00
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The Type-it Mutation Detect PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The Type-it Mutation Detect PCR Kit is especially designed to enable fast and reliable detection of mutations such as deletions, insertions, and translocations. The kit is based on highly specific HotStarTaq Plus DNA Polymerase and a patented buffer system, both of which enable reliable amplification of mutant loci by multiplex PCR without optimization. Subsequent analysis is straightforward and easy and can be carried out on agarose gels, automated electrophoresis instruments, and also by high-resolution capillary sequencing.

Performance

The Type-it Mutation Detect PCR Kit outperformed kits tested from other suppliers and ensures reliable analysis of mutations. The kit is specifically developed and functionally validated for multiplex PCR-based analysis of mutations such as deletions or insertions and detection of genetically modified organisms or microbes (see figure " Sensitive detection of a mutated cancer-related gene"). The kit is also suitable for use as a preamplification method for commercially available SNP detection PCR-based systems such as the SNaPshot system provided by Applied Biosystems (see figure " Superior preamplification of SNPs").
See figures

Principle

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.

Q-Solution

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.

CoralLoad Dye

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.

See figures

Procedure

The Type-it Mutation Detect PCR Kit includes dedicated, application-specific protocols, optimized for simultaneous amplification and subsequent detection of loci carrying mutations or SNPs to ensure reliable results for routine analysis or establishment of new assays. Reactions can be set up at room temperature, ensuring greater convenience and ease of use.

Fast and simple way to reliable genotyping results

The Type-it Mutation Detect PCR Kit enables fast and easy multiplex assay development for accurate and reproducible genotyping results. Whether its detection of translocations, deletions, insertions, or preamplification of genomic loci for SNP analysis methods such as the SNaPshot (Applied Biosystems), just add the template and primers, and start the thermal cycler program according to the optimized protocol. The reaction mixture contains all of the reagents required for mutation-specific multiplex PCR and enables accurate results without the need for lengthy optimization procedures, when compared with current methods (see figure " Successful multiplex PCR-based genotypic analysis"). 

Time and cost savings due to straightforward procedure

Some studies require analysis of a large number of different mutations of a certain gene related to a disease (e.g., deletions, translocations, or insertions) requiring a large number of PCR reactions when performing  singleplex or low-plex grade PCR analysis, thereby leading to increases in both costs and analysis time. The Type-it Mutation Detect PCR Kit ensures comparable amplification efficiency for all amplicons in a high-plex grade multiplex experiment and allows more mutation targets to be combined ensuring significant time and cost savings (see figure  Sensitive detection of a mutated cancer-related gene).

Reliable preamplification of SNPs

The Type-it Mutation Detect PCR Kit is also highly suited for multiplex PCR-based preamplification of SNPs (e.g., the SNaPshot system from Applied Biosystems). The kit outperformed kits tested from other suppliers and consistently delivers reliable results, without the need for tedious optimization procedures (see figure " Superior preamplification of SNPs").

See figures

Applications

The Type-it Mutation Detect PCR Kit is used to analyze mutations, deletions, insertions, duplications, translocations, and for SNP preamplification (e.g., SNaPshjot Multiplex Kit) and is used in diverse research fields such as:

  • Typing of disease loci
  • GMO analysis
  • Typing of transgenic plants or animals

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsMultiplex PCR, detection of mutations, preamplification of SNPs
mastermixYes
reactiontypePCR amplification
productuseFunctionally validated and developed for reliable mutation analyis
realtimeorendpointEndpoint
sampletargettypeGenomic DNA
singleormultiplexMultiplex
withwithouthotstartWith

Resources

Brochures & Guides (2)
Second edition — innovative tools
Addressing critical factors and new solutions
Kit Handbooks (1)
For optimization-free multiplex PCR-based mutation detection or preamplification of SNPs

FAQ

What is the advantage of using the Type-it Mutation Detect PCR Kit?

The Type-it Mutation Detect PCR Kit was specifically developed and validated for the detection of mutations such as deletions, insertions, and translocations. It is also highly suited for multiplex PCR-based preamplification of SNPs as preparation for genotyping systems like ABI's SNaPshot. Dedicated protocols for various detection platforms (Agarose gels, QIAxcel System, Agilent BioAnalyzer, and Capillary Sequencers) are available in the Type-it Mutation Detect PCR Handbook.

 

 

FAQ ID -2064
Why is CoralLoad included in the Type-it Mutation Detect, but not in the Type-it Microsatellite PCR Kit?

CoralLoad Dye unfortunately interferes with Capillary Sequencers and increases the risk of damaging the capillaries of these detection platforms. It is not included in the Type-it Microsatellite PCR Kit, since microsatellite loci are analyzed predominantly on Capillary Sequencers due to their high-resolution capability.

By comparison, mutations are very often analyzed on agarose gels, or the QIAxcel System, compatible with CoralLoad dye included in the Type-it Mutation Detect PCR Kit.

 

FAQ ID -2068
Are there recommendations for sensitive detection of underrepresented fragments using the Type-it Mutation Detect PCR Kit?

Yes, special recommendations for detecting underrepresented fragments with the Type-it Mutation Detect PCR Kit can be found in Appendix E of the Type-it Mutation Detect PCR Handbook.

 

 

FAQ ID -2066
Why is the Type-it Mutation Detect PCR Kit recommended for preamplification of SNPs?

Preamplification of SNPs in preparation for genotyping systems like Applied Biosytem's SNaPshot requires single PCRs to be amplified and pooled, before input into this system. Using the Type-it Mutation Detect PCR Kit, all fragments can be obtained at once saving time and costs. Please follow the protocol 'Amplification of Mutations (Detection on Agarose Gels or the QIAxcel System or Agilent 2100 Bioanalyzer)' in the Type-it Mutation Detect Handbook.

 

 

 

 

FAQ ID -2067
Can the Type-it Mutation Detect PCR kit be used for multiplex PCR of fragments longer than 500 bp?

Yes, the Type-it Mutation Detect PCR kit can be used for fragments >500 bp by following the recommendations in the Type-it Mutation Detect PCR Handbook (increase extension time by 30 seconds for each additional 0.5 kb).

 

 

 

FAQ ID -2065