Type-it HRM PCR Kit

应用高分辨率熔解（HRM）分析快速、准确的检测基因突变和SNPs

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Type-it HRM PCR Kit (400)

Cat. No. / ID: 206544

For 400 x 25 μl reactions: 4 x 1.3 ml of 2x HRM PCR Master Mix (contains HotStarTaq Plus DNA Polymerase, EvaGreen Dye, optimized concentration of Q-solution, dNTPs, and MgCl₂) and RNase-Free Water

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Reactions

400

2000

Type-it HRM PCR Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

准确、可靠的检测细微的序列变化
非常适合用于具有HRM功能的PCR仪
新型的EvaGreen荧光染料有助于获得清晰的熔解曲线
便于开发新的可靠的HRM基因分型方案
使用方便的预混液和经优化的操作流程

Product Details

Type-it HRM PCR Kit提供可靠的解决方案，用于通过HRM分析进行快速、准确的基因分型。该试剂盒经优化，可成功分析难扩增的基因组位点。该试剂盒非常适合突变扫描，可用于筛选样本的未知突变。可简便的建立新的HRM基因分型检测。该试剂盒可稳定的获得特异性的扩增产物，减少非特异性扩增，获得可靠结果，无需进行耗时的PCR参数优化。这使得实验标准化、灵活，大大节省时间和成本。该试剂盒可配合多种具有HRM功能的real-time PCR仪使用，如Rotor-Gene Q实时荧光定量PCR分析仪、Rotor-Gene 6000、LightCycler 480和Applied Biosystems 7500 Fast System。

Performance

Type-it HRM PCR Kit经验证可准确分辨序列突变，非常适合使用新型的HRM技术明确等位基因分型。通过HRM技术，之前未知的和复杂的序列变化被认为是具有挑战性的基因分型应用，现在都可轻松分析（参见" Highly accurate genotyping"）。

可靠、准确地检测细微的序列突变

试剂盒中提供的预混液含有新型的双链DNA结合荧光染料EvaGreen，还包括经优化的HRM缓冲液、HotStarTaq Plus DNA Polymerase、Q-Solution和dNTPs。这些组分确保PCR的特异性，提供可靠的结果，甚至用于难处理的基因组位点。Type-it HRM PCR Kit优于其他供应商的试剂盒，可扩增有挑战性的细微序列突变。与其他商业化HRM预混液化合组分相比，Type-it HRM PCR Buffer的独特组分确保高度严谨和定向的引物结合（参见 " Highly specific and successful amplification of difficult genomic loci"）。与其他供应商的试剂盒不同，Type-it HRM PCR Kit可在多种real-time仪器上获得持续稳定的表现，确保对Class IV SNPs（参见" Successful genotyping of an A/T Class IV SNP"）和基因突变（参见 " Successful typing of gene mutations"）进行高度准确地检测。由于减少了需要重测的样本数量，可大大节省时间和成本。

快速、简单的突变扫描

Type-it HRM PCR Kit可进行高度可靠的突变扫描。未知基因突变（插入和缺失）可被轻松检测并被可靠区分，产生不同的熔解曲线（参见" Successful mutation scanning"）。

See figures

Highly accurate genotyping.

Highly specific and successful amplification of difficult genomic loci.

Successful genotyping of an A/T Class IV SNP.

Successful typing of gene mutations.

Successful mutation scanning.

Principle

HRM是一种封闭管式的、PCR后的分析方法，引起了极大地科学兴趣。HRM可根据分离（熔解）行为对双链PCR产物进行描述，随着温度升高，双链DNA（dsDNA）就会转变成单链DNA（ssDNA）。PCR产物可根据序列、长度、GC含量或链互补性，甚至是单个碱基变化进行区分。之前未知的和复杂的序列变化被认为是具有挑战性的基因分型应用，现在都可轻松分析（参见" Highly accurate genotyping"）。与常规方法不同，HRM可避免PCR产物的残留污染，是一种更简单、更经济的基于探针的基因分型分析。

独特的试剂盒组成确保高度特异性的扩增（见表）。

新型的EvaGreen染料，用于不同的熔解曲线

Type-it HRM PCR Kit含有的EvaGreen是第三代饱和荧光染料，可选择性地结合到双链DNA上。与常规的SYBR^® Green I相比，EvaGreen可使用更高浓度，无PCR抑制，对GC含量丰富和AT含量丰富的区域具有相同的结合能力，无明显的序列偏向性。这使得EvaGreen非常适合对多种类型的PCR产物进行HRM分析，对更低的荧光差异产生不同的熔解曲线，确保获得标准化结果。

优化的HRM PCR Master Mix

HRM PCR Master Mix由HotStarTaq Plus DNA Polymerase和一种新型的HRM PCR缓冲液体系组成，能得到高度特异性的扩增和不同的熔解曲线，可用于难处理得基因组位点，如Class IV SNPs（参见" Successful genotyping of an A/T Class IV SNP"）。确保目的PCR产物的特异性扩增；减少非特异性产物和引物二聚体的形成，如果没有去除（参见" Successful typing of gene mutations"）。

新型的缓冲液（包含在预混液中）通过在每个PCR循环的退火步骤中提高特异性引物的结合比率来维持每个PCR循环的特异性扩增。KCl 和 (NH₄)₂SO₄独特的平衡组合，使得该缓冲液在很宽的退火温度范围和Mg²⁺浓度范围内提供严格的引物退火条件。无需通过改变退火温度和Mg²⁺浓度来优化PCR反应。

适合难处理的突变位点

Type-it HRM PCR Kit是一种简单检测难处理突变位点的有力体系（参见" Highly specific and successful amplification of difficult genomic loci"）。位于GC含量高或高级二级结构区域的突变或SNPs都难以扩增，用于常规的HRM PCR方法。2x HRM PCR Master Mix包含新型的PCR添加剂Q-Solution，使用特定浓度的Q-Solution可帮助克服这些困难。Q-Solution通过修饰DNA的熔解行为，可成功扩增难处理的目标序列，无需优化。

Type-it HRM PCR Kit的特点
组成	优点
2x HRM PCR Master Mix	建立新的检测无需优化专门用于突变和SNPs的HRM分析专门用于适合HRM分析的所有循环仪方便的反应体系构建，减少移液误差
HotStarTaq Plus DNA Polymerase	高度特异性扩增室温下快速、简单的构建反应体系
Type-it HRM PCR Buffer	不同的熔解曲线提高扩增特异性
Q-Solution	提高对难处理模板的扩增能力
EvaGreen Dye	新型的饱和dsDNA结合染料不同的熔解曲线分析

See figures

Highly accurate genotyping.

Successful genotyping of an A/T Class IV SNP.

Successful typing of gene mutations.

Highly specific and successful amplification of difficult genomic loci.

Procedure

Type-it HRM PCR Kit包括专门的、应用特异性的操作流程，预优化后用于多种real-time循环仪，如Rotor-Gene Q实时荧光定量PCR分析仪、Rotor-Gene 6000和LightCycler 480。此外，采用试剂盒提供的优化操作流程还可使用Applied Biosystems 7500 Fast System和Applied Biosystems 7900。我们的网站上提供详细的操作流程。

快速循环步骤，易构建的HRM检测，可直接获得结果

Type-it HRM PCR Buffer的独有特色和试剂盒提供的优化操作流程，确保一次获得成功的HRM基因分型结果。无需冗长的反应参数优化，新的HRM基因分型检测可快速、简单地标准化，并用于常规研究（参见" Successful HRM analysis without the need for optimization"）。此外，Type-it HRM PCR Kit的快速循环操作流程通过缩短PCR的运行时间提高通量，可更快获得准确的结果。

方便的试剂盒规格

该试剂盒采用即用型、预优化预混液的规格，更加方便。使用预混液可节约时间，简化反应体系构建的手工操作，通过排除移液误差和污染的可能来源提高可重复性，尽量减少移液步骤，无需繁琐的计算。使用预混液可进行室温下的反应体系构建，快速、简单。预混液中含有的HotStarTaq Plus DNA Polymerase在95°C，5-minute被激活，可在热循环程序中简单设置。

See figures

Successful HRM analysis without the need for optimization.

Applications

Type-it HRM PCR Kit可用于多种应用，包括：

检测插入、缺失和异位
扫描基因突变
SNP基因分型
检测和区分微生物突变体

Type-it HRM PCR Kit是多种研究领域的通用工具，包括：

疾病和癌症基因位点的分型
生物标记物的发现
疾病相关研究
转基因植物或动物的分型
病原体检测和基因分型

Supporting data and figures

Successful typing of gene mutations.

[A] Difference plot showing reliable discrimination between the wild-type sequence (blue), the c.35 G>C mutation (brown), and the c.37G>T mutation (pink). The Type-it HRM PCR Kit ensured reproducible discrimination with high confidence — without the need for optimization. [B] The c.35 G>C mutation could not be resolved from the wild-type even after extensive optimization of Mg²⁺ concentration and cycling parameters when using the HRM master mix from Supplier R. The mutation affects the amino acid in position 12, resulting in p.G12A and in position 13, resulting in p.G13C.

Specifications

Features	Specifications
Applications	Detection of SNPs, mutations and mutation scanning
Product use	Functionally validated and developed for reliable detection of genetic differences using HRM
Reaction type	PCR amplification
Enzyme activity	5'-> 3' Exonuclease activity
With or without ROX	Without ROX
Sequence specific Probe	Not necessary, EvaGreen dye for detection included in the Mastermix
Sample/target type	Genomic DNA
With/without hotstart	With
Mastermix	Yes
Real-time or endpoint	Both

Resources

Second edition — innovative tools

Addressing critical factors and new solutions

Download Safety Data Sheets for QIAGEN product components.

Second edition — innovative tools

Addressing critical factors and new solutions

FAQ

The Type-it HRM PCR Kit uses EvaGreen as this is a dsDNA saturating dye enabling more distinct melting curves for High-resolution melting experiments compared to SYBR Green dye.

FAQ ID -2196

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

Minimal template variability from sample to sample is essential for reliable HRM analysis with the Type-it HRM PCR Kit. Traces of salts or varying elution buffers will influence the melting behaviour of the amplified DNA. Therefore, an identical extraction procedure for all samples should be selected.

FAQ ID -2197

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

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

A deletion or insertion of more than 2 base pairs can be usually detected in a background of 85-95% wild-type DNA using the Type-it HRM PCR Kit.

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

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