Type-it Fast SNP Probe PCR Kit

For accurate and reliable SNP genotyping using TaqMan® or TaqMan® MGB probes

S_1084_5_GEN_V2

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Type-it Fast SNP Probe PCR Kit (800)

Cat. No. / ID:  206045

For 800 x 25 μl reactions: 6 x 1.7 ml of 2x Type-it Fast SNP Probe PCR Master Mix, 5x Q-Solution, RNase-Free Water
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A$1,479.00
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4000
The Type-it Fast SNP Probe 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

  • Validated using TaqMan® SNP Genotyping Assays
  • Automated allele calling and tight fluorescence clusters
  • Suitable for difficult SNP loci or very low template amounts
  • Up to 40% time savings due to fast cycling procedure

Product Details

The Type-it Fast SNP Probe PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a newly developed buffer system, both of which enable reliable and clear allelic discrimination. The combination of all components provided in the master mix result in improved accuracy and highly specific probe binding. The Type-it Fast SNP Probe PCR Kit is validated using TaqMan® SNP Genotyping Assays and enables reproducible SNP genotyping — even with difficult SNP loci (e.g., GC rich) or low amounts of starting template.

Performance

The Type-it Fast SNP Probe PCR Kit consistently ensures highly accurate SNP genotyping. 

Highly stringent and specific binding of the allele-specific probe 

The Type-it Fast SNP Genotyping PCR Master Mix is based on highly specific HotStarTaq Plus DNA Polymerase and a newly developed SNP genotyping PCR buffer system, both of which enable highly specific probe binding and consistently strong fluorescent signals. Compared to other commercially available SNP genotyping master mix chemistries, wider and clearer separation of allele clusters is obtained (see figure " Highly specific probe binding").

High call rates even for low template amounts

Even with challenging targets or difficult SNP loci, the Type-it Fast SNP Probe PCR Kit provides reliable SNP genotyping — well-separated allele clusters and tight clustering of identical alleles are achieved, resulting in high call rates  (see figure " Successful automated allele calling"). The Type-it Fast SNP Probe PCR Kit outperformed kits tested from other suppliers, ensuring tight clustering of alleles — even with 1 ng of template DNA (see figure " Reliable SNP genotyping with small amounts of template").

See figures

Principle

The Type-it Fast SNP Probe PCR Kit is available in a convenient master mix format consisting of highly specific HotStarTaq Plus Polymerase and a unique SNP genotyping buffer system designed for fast and efficient amplification. The unique kit components provide tight allele clustering and outstanding separation to ensure high call rates, enabling reproducible and accurate results (see table).

High specificity and sensitivity

HotStarTaq Plus DNA Polymerase, provided in the master mix, which ensures highly specific amplification, even with low template amounts. HotStarTaq Plus DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer dimers at low temperatures.

Unique buffer system

The Type-it Fast SNP PCR Buffer, also included in the master mix, is specifically designed for fast-cycling SNP genotyping using sequence specific 5'-nuclease probes. The unique composition of the Type-it Fast SNP PCR Buffer ensures highly stringent and specific binding of the allele-specific probe (match probe). This is due to the altered melting behaviour of the probes resulting in a narrower probe melting temperature window. Based on the original QIAGEN PCR Buffer, this innovative formulation enables a high ratio of specific-to-nonspecific primer binding during the short annealing step of every PCR cycle. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR 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 dramatically reduced and often not required.

Additives in the Type-it Fast SNP PCR buffer, such as Q-Solution, provide reaction conditions for amplification of difficult genomic regions and difficult SNP loci. Innovative Q-Bond technology (for fast cycling) allows SNP genotyping results to be achieved faster (see figure " Mechanism of fast cycling during annealing"). The master mix also contains ROX dye at a concentration compatible with SNP genotyping on all instruments from Applied Biosystems. However, ROX dye is also compatible with other instruments not requiring ROX as a passive reference dye (see table).

Kit features
Kit contents Features
2x Master Mix format* Developed for SNP genotyping with TaqMan® MGB probes
Suitable for use with all cyclers suitable for SNP genotyping
HotStarTaq Plus DNA Polymerase Fast and easy reaction setup at room temperature
Highly specific amplification, even with low template amounts
Type-it Fast SNP Probe PCR Buffer Wide separation of genotype clusters
Tight allele clusters and high allele calling rates
Increased specificity in probe binding
Fast cycling due to Q-Bond Molecule
Q-Solution Tighter clusters and higher signals in scatter plot analysis for highly GC-rich SNP loci
Can further improve suboptimal allele calling
See figures

Procedure

The Type-it Fast SNP Probe PCR Kit is functionally verified with commercially available SNP genotyping assays and is compatible with TaqMan® MGB Probes, as well as user-developed probe-based assays consisting of TaqMan® MGB, TaqMan®, or other dual-labeled probes. The kit includes streamlined, preoptimized protocols for fast and reliable analysis.

Fast-cycling procedure for straightforward results

The Type-it Fast SNP Probe PCR Master Mix provides reaction conditions for fast-cycling PCR using sequence-specific probes. The buffer includes proprietary Q-Bond Molecule, which allows short cycling times on standard cyclers and on fast cyclers with rapid ramping rates. The fast-cycling procedure increases throughput by reducing PCR run times by up to 40% (see figure " Genotyping workflow").

Convenient kit format

The kit is provided in a ready-to-use, preoptimized master mix format 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. HotStarTaq Plus DNA Polymerase (included in the master mix) is activated by a 5-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. Room-temperature reaction setup using the master mix is fast and easy.

Suitable instruments

The Type-it Fast SNP Probe PCR Kit is optimized for amplification of SNP PCR assays on any suitable standard and fast ramping cycler compatible with optical PCR plates that fit into real-time PCR instruments used for fluorescence plate read analysis (see table).

Suitable instruments
Cyclers Model
Real-time PCR cyclers QIAGEN: Rotor-Gene Q
ABI PRISM 7900 (all series)
ABI StepOne and StepOne Plus
Applied Biosystems 7500 (all series)
Applied Biosystems ViiA 7
Bio-Rad: iCycler iQ
Bio-Rad: CFX series
Roche: LightCycler 480
Roche: LightCycler Nano
Agilent: Mx3000P and Mx3005P
Standard cyclers All cyclers in standard-ramping modes (e.g., GeneAmp 9700)
All cyclers in fast-ramping modes (e.g., GeneAmp 9800)
See figures

Applications

The Type-it Fast SNP Probe PCR Kit is used to detect SNPs using TaqMan® MGB probes and can be used in various fields of research, including:

  • Typing of disease loci
  • Biomarker discovery

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsProbe-based SNP Genotyping
Product useFunctionally validated and developed for reliable SNP Genotyping
MastermixYes
Real-time or endpointBoth
With or without ROXROX included in Master Mix
With/without hotstartWith
Sample/target typeGenomic DNA
Enzyme activity5'-> 3' Exonuclease activity
Sequence specific ProbeTaqMan® Genotyping Assays, TaqMan® or TaqMan® MGB probes
Reaction typePCR amplification

Resources

Brochures & Guides (3)
Second edition — innovative tools
Now with even more applications!
Addressing critical factors and new solutions
Kit Handbooks (1)
Certificates of Analysis (1)

FAQ

How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

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
Can other probe technologies besides TaqMan® be used with the Type-it Fast SNP Probe PCR Kit?

The Type-it Fast SNP Probe PCR Kit was developed and validated for use with 5'-3' nuclease (hydrolysis) probes like TaqMan® MGB or standard TaqMan® probes. We have not yet tested other probe technologies with this kit.

See trademarks

FAQ ID -2058
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

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
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

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
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

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
What do I do if no fluorescent signal is detected in a real-time PCR assay?

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
How do I select appropriate reporter and quencher combinations for multiplex PCR?

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
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

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
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. 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
How do I ensure reliable results for High Resolution Melting (HRM) assays?

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 CT values are below 30 and differ no more than 3 CT 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
Why is special Type-it Fast SNP Probe PCR chemistry required for TaqMan® SNP Genotyping?

SNP Genotyping using TaqMan® probes is a unique application requiring the development of a unique reaction chemistry. The Type-it Fast SNP Probe PCR Kit allows wide separation of genotype clusters and reliable amplification of any difficult genomic region for high call rates.

See trademarks.

FAQ ID -2057
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

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 (Tm) 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 Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096