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Rotor-Gene Type-it HRM Discovery Kit

For demonstration and evaluation of successful HRM-based genotyping analysis on the Rotor-Gene Q
  • Successful evaluation of HRM-based genotyping on the Rotor-Gene Q
  • Includes 2 preoptimized HRM assays (class IV A/T SNP and deletion)
  • Complete kit format with master mix, assays, and template DNA
  • Distinct melting curves due to novel EvaGreen fluorescent dye

The Rotor-Gene Type-it HRM Discovery Kit has been developed to demonstrate the high performance of Type-it HRM chemistry, in combination with the advanced thermal and optical properties of Rotor-Gene Q, for detection of gene mutations and SNPs. The reliability and reproducibility of high-resolution melting (HRM) analysis with Rotor-Gene technologies is thoroughly evaluated using the Rotor-Gene Type-it HRM Discovery Kit. For a wealth of information on this unique technology and its successful utilization in a range of research fields, please visit our HRM Spotlight.
The Rotor-Gene Type-it HRM Discovery Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Deletion assay.
Expected results of the 9 bp deletion assay are shown. Red: WT (sample 6); blue: 100% mutant (sample 7); green: 50% mutant (sample 8); grey: 20% mutant (sample 9).
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Class IV SNP assay.
Expected results of the class IV SNP (A to T transversion) assay are shown. Red: WT (sample 1); blue: mutant (sample 2); green: heterozygous (sample 3).
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Successful genotyping of an A/T Class IV SNP.
Typing the SNP (rs2270938) in the human GYS1 gene using the Type-it HRM PCR Kit results in highly reproducible and accurate results, but a strong variation is observed when using the master mix from Supplier QIII. [A] Normalized melting curve and difference plot showing successful and reliable discrimination of all 3 genotypes (wild-type, heterozygote, and mutant) of a Class IV SNP using the Type-it HRM PCR Kit. [B] Wild-type and mutant of the same locus could not be resolved when performing the same experiment using the kit from Supplier QIII, resulting in unsuccessful genotyping. [A] and [B] Blue: wild-type; Green: heterozygous; Red: mutant.
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Highly specific and successful amplification of difficult genomic loci.
[A] Type-it HRM PCR Buffer ensures highly specific amplification even of difficult genomic loci (Human ENG gene, Exon 11, 356 bp fragment, 64% GC). Only the expected specific PCR products are obtained. [B] In contrast, using the kit from Supplier R results in nonspecific products, even with prior optimization of Mg2+ and cycling parameters.
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Highly accurate genotyping by HRM analysis.
HRM analysis of an A/C Class II SNP (rs405509) in the human ApoE gene was performed using the Type-it HRM PCR Kit on the Rotor-Gene Q real-time PCR instrument. HRM analysis of the indicated genotypes is shown as either [A] a standard normalized melt curve or [B] a difference plot.
Performance
The Rotor-Gene Type-it HRM Discovery Kit allows evaluation of HRM-based genotyping analysis on the Rotor-Gene Q, using Type-it HRM chemistry. The kit enables successful detection of a 9 bp gene mutation (see figure Deletion assay) and/or a class IV SNP (A to T transversion) (see figure Class IV SNP assay) via high-resolution melting (HRM) analysis.
 
High-performance HRM on the Rotor-Gene Q
The Rotor-Gene Q is a real-time PCR instrument that is highly suitable for HRM due to its outstanding thermal and optical uniformity. Features such as a specially tuned high-intensity HRM channel, comprehensive analysis software, and high data acquisition rates, make the Rotor-Gene Q uniquely suited for even the most challenging HRM applications. The Rotor-Gene Q is the capable of easily discriminating even the most difficult class IV A/T SNPs with melting point differences below 0.2°C (see figure Powerful data analysis enables identification of a class IV SNP).
 
Highly specific amplification
The preoptimized 2x HRM PCR Master Mix provided with the Rotor-Gene Type-it HRM Discovery Kit consists of HotStarTaq Plus DNA Polymerase and an innovative HRM PCR buffer system, both of which enable highly specific amplification and distinct melting curves, even with difficult genomic loci such as class IV SNPs (see figures Successful genotyping of an A/T class IV SNP, Powerful data analysis enables identification of a class IV SNP, and Class IV SNP assay). Specific amplification of the desired PCR product is ensured and generation of nonspecific products and formation of primer dimers is minimized, if not eliminated (see figure Highly specific and successful amplification of difficult genomic loci). The master mix included with the kit also contains EvaGreen dye for distinct melting curves.
Principle
High-resolution melting (HRM) is a closed-tube, post-PCR analysis method that has raised enormous scientific interest and has utility in diverse applications such as genotyping, epigenetics, and pathogen typing. HRM characterizes double-stranded PCR products based on their dissociation (melting) behavior as they transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) with increasing temperature. PCR products can be discriminated according to sequence, length, GC content, or strand complementarity, down to single base pair changes. Previously unknown and even complex sequence variations, as seen in challenging genotyping applications, can be readily detected and characterized in an easy and straightforward manner (see figure Highly accurate genotyping by HRM analysis). HRM is easier and more cost-effective than probe-based genotyping analysis and unlike conventional methods, it prevents carry-over post-PCR contamination.

HRM can be easily performed using the Rotor-Gene Q for applications such as:
  • SNP genotyping
  • Mutation detection
  • Pathogen typing
  • Methylation analysis

Predesigned assays for effortless evaluation of HRM technology
The Rotor-Gene Type-it HRM Discovery Kit demonstrates the detection of deletions or SNPs via high-resolution melting analysis. HRM technology enables rapid characterization of DNA samples based on their melting behavior following PCR amplification. The Rotor-Gene Type-it HRM Discovery Kit has been developed to exhibit the superior thermal and optical capabilities of the Rotor-Gene instrument in conjunction with the exceptional performance of the HRM PCR Master Mix. In addition to a master mix and template DNA, the kit provides 2 preoptimized HRM assays — a class IV A/T SNP (see figure Class IV SNP assay) and a deletion assay (see figure Deletion assay). Additionally, manual pipetting steps can be avoided by using the QIAgility, a compact bench-top instrument that provides rapid, high-precision PCR setup.
 
Novel EvaGreen dye for distinct melting curves
EvaGreen, a third-generation, saturating fluorescent dye that selectively binds to double-stranded DNA is included in the 2x HRM PCR Master Mix. In contrast to conventional SYBR® Green I, EvaGreen can be used at higher concentrations without PCR inhibition and shows equal binding affinity for GC- and AT-rich regions, with no apparent sequence preference. This makes EvaGreen highly suited to HRM analysis of all types of PCR products, enabling distinct melting curves due to visualization of lower fluorescent differences, thereby ensuring standardized results.
 
Preoptimized HRM PCR master mix
The 2x HRM PCR Master Mix consists of HotStarTaq Plus DNA Polymerase and an innovative HRM PCR buffer system. The stringent hot start provided by chemically modified HotStarTaq Plus DNA Polymerase allows reaction setup at room temperature. The unique composition of the HRM PCR Buffer ensures extremely stringent and targeted primer annealing, leading to highly specific amplification.
Procedure
The Rotor-Gene Type-it HRM Discovery Kit allows both manual and automated reaction setup. The kit handbook provides 2 preoptimized protocols for convenient reaction setup and a protocol for HRM PCR on the Rotor-Gene Q. The protocol for manual reaction setup includes pipetting schemes for both assays. The automated reaction setup protocol allows high-precision reaction setup using the QIAgility. Preoptimized cycling conditions are included in the protocol for HRM PCR.
Applications
The successful use of the Rotor-Gene Q and Type-it HRM chemistry for HRM-based genotyping applications such as SNP or mutation detection is easily demonstrated using the Rotor-Gene Type-it HRM Discovery Kit.
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