Type-it Microsatellite PCR Kit

For fast and reliable multiplex PCR analysis of microsatellite loci

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Type-it Microsatellite PCR Kit (200)

Cat. No. / ID: 206243

For 200 x 25 μl reactions: Type-it Multiplex PCR Master Mix (3 x 0.85 ml), 5x Q-Solution (1 x 2 ml), and RNAse-Free Water (2 x 1.9 ml)

DKK 2,310.00

Reactions

200

2000

Quantity

The Type-it Microsatellite PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

Reliable microsatellite analysis by multiplex PCR
Microsatellite assay development without optimization
Successful and specific coamplification of all fragments
Optimized protocol for fast and reliable results
Easy instructions for use with various downstream analysis platforms

Product Details

The Type-it Microsatellite PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a patented buffer system, both of which enable reliable multiplex PCR-based microsatellite analysis without optimization. The combination of all components provided in the master mix and the specialized formulation result in highly specific amplification of all loci in parallel. The kit is dedicated to fast and reliable microsatellite analysis by multiplex PCR. Optimized protocols are also provided to enable subsequent analysis either by high-resolution capillary sequencing or by using other electrophoresis instruments.

Performance

The Type-it Microsatellite PCR Kit outperformed kits tested from other suppliers and ensures reliable microsatellite analysis. The kit is specifically developed and functionally validated for multiplex PCR-based analysis of microsatellites or minisatellites such as STRs or VNTRs used, for example, in relationship analysis or population genetics (see figure "").

See figures

Reliable 13-plex STR analysis without the need for optimization.

Principle

The Type-it Microsatellite PCR Kit provides fast and reliable genotyping analysis of humans, animals, plants, and bacteria using microsatellites, STR, or VNTR markers, without the need for lengthy optimization procedures. The Type-it Microsatellite PCR Kit is based on proprietary QIAGEN multiplex technology and includes highly specific HotStarTaq Plus DNA Polymerase, a chemically modified hot-start enzyme, and a specially developed multiplex buffer system including Factor MP. Together, these components ensure high yields and enable reliable multiplex amplification of all microsatellite loci of interest. The kit includes dedicated protocols for the amplification of microsatellites or STRs, 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.

Type-it Microsatellite PCR Buffer

Unique Type-it Microsatellite PCR Buffer facilitates the amplification of multiple PCR products. In contrast to conventional PCR reagents, the Type-it Microsatellite 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 Microsatellite PCR Kit includes 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.

See figures

Stable and efficient annealing.

Procedure

Dedicated, application-specific protocols, optimized for analysis of microsatellites on high-resolution capillary sequencers, are included with the Type-it Microsatellite PCR Kit to ensure reliable results for routine analysis, as well as for the 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 Microsatellite PCR Kit enables fast and easy multiplex assay development for accurate and reproducible genotyping results. Whatever the application — microsatellite, STR, SSR, or VNTR analysis — 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 microsatellite-specific multiplex PCR and enables accurate results without the need for lengthy optimization procedures compared to current methods (see figure " Successful multiplex PCR-based genotyping analysis").

Optimized for use with capillary sequencers

Microsatellite analysis on high-resolution detection systems such as capillary sequencers is often associated with the problem of uneven product yield and huge intensity differences of fluorescent signals. The Type-it Microsatellite PCR Kit overcomes this limitation and ensures high product yields for all amplicons in a multiplex experiment. The kit includes optimized protocols for use with fluorescent primers and outperforms enzyme solutions from other suppliers (see figure " Reliable 13-plex STR analysis without the need for optimization").

See figures

Successful multiplex PCR-based genotyping analysis.

Reliable 13-plex STR analysis without the need for optimization.

Applications

The Type-it Microsatellite PCR Kit is used to analyze microsatellites, STRs (short tandem repeats), SSRs (simple sequence repeats), and VNTRs (variable number of tandem repeats) and can be used in diverse research fields such as:

Species or individual identification
Analysis of relationship
Microsatellite instability
Population genetics

Supporting data and figures

Reliable 13-plex STR analysis without the need for optimization.

Only 2 out of 4 channels (FAM and PET) of a 3730 xl Capillary Sequencer (ABI), representing 6 of 13 analyzed STR loci are shown. [A] Using the Type-it Microsatellite PCR Kit under standard conditions, all 6 STR loci are reliably amplified. [B] Despite the increased number of cycles and preoptimized PCR conditions for the hot-start method of Supplier A — compared with 25 cycles and standard conditions for the Type-it Microsatellite Kit — several specific peaks are missing and nonspecific signals are observed (arrows).

Specifications

Features	Specifications
Applications	Multiplex PCR, Microsatellites, STRs, VNTRs, SSRs
Reaction type	PCR amplification
Product use	Functionally validated and developed for reliable Microsatellite Analysis
Mastermix	Yes
Real-time or endpoint	Endpoint
Sample/target type	Genomic DNA
Single or multiplex	Multiplex
With/without hotstart	With

Resources

Second edition — innovative tools

Addressing critical factors and new solutions

For optimization-free and reliable multiplex PCR based analysis of microsatellites

FAQ

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

FAQ ID -9093

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

The Type-it Microsatellite PCR Kit was optimzed and validated for this size range since microsatellite analysis typically only uses fragments of up to 500 bp in length.

FAQ ID -2060

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

FAQ ID -90990

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line. Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

FAQ ID -9094

All capillary sequencers can be used for detection of PCR product amplified with the Type-it Microsatellite PCR Kit.

FAQ ID -2063

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098

Yes. We tested the Type-it Microsaltellite PCR Kit on different commonly used fast and normal PCR cyclers. No significant differences in performance were observed with the systems tested.

FAQ ID -2062

Microsatellite analysis on high resolution detection systems such as capillary sequencers is often associated with the problem of uneven product yield and huge intensity differences of fluorescent signals. The Type-it Microsatellite PCR technology was specifically developed and validated to overcome this limitation and ensure high product yields for all amplicons up to 500 bp long in a multiplex experiment. The kit includes optimized protocols for use with fluorescent primers and outperforms enzyme solutions from other suppliers.

FAQ ID -2059

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.

FAQ ID -9099

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097

For established systems, previously optimized annealing temperatures can be used with the Type-it Microsatellite PCR Kit. For new PCR systems, follow the guidelines in Appendix B, 'Design of Multiplex Primers', in the Type-it Microsatellite PCR Handbook.

FAQ ID -2061

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096