应用高分辨率熔解(HRM)分析快速、准确的检测基因突变和SNPs

  • 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

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。

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Type-it HRM PCR Kit (100)
For 100 x 25 μl reactions: 1 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
206542
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Type-it HRM PCR Kit (400)
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
206544
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Type-it HRM PCR Kit (2000)
For 2000 x 25 μl reactions: 25 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
206546
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The Type-it HRM PCR 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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Successful typing of gene mutations.|Highly accurate genotyping.|Successful genotyping of an A/T Class IV SNP.|Highly specific and successful amplification of difficult genomic loci.|Successful mutation scanning.|Successful HRM analysis without the need for optimization.|
[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.|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. The Type-it HRM PCR Kit ensured highly accurate HRM analysis.|Typing the SNP (rs2270938) in the human GYS1 gene using the Type-it HRM PCR Kit results in highly reproducible and accurate results, but 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.|[A] The Type-it HRM PCR Buffer ensures high specificity in the 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, the kit from Supplier R results in nonspecific products, even with prior optimization of Mg2+ and cycling parameters.|Gene mutations (insertions and deletions in EGFR exon 19) were analyzed using the Type-it HRM PCR Kit on the Rotor-Gene Q. HRM analysis of the indicated genotypes is shown as a difference plot. Analysis was performed on the Rotor-Gene Q 5plex HRM cycler. WT: EGFR Exon 19 wild-type sequence, mut1: c.2235_2249del15, mut3: c.2237_2252del16insT, c.2237_2238ins18, mut4: c.2237_2238ins18, mut5: c.2239_2248del10insC, mut6: c.2240_2254del15, mut7: c.2240_2257del18.|The Type-it HRM PCR Kit provides an easy procedure for reliable genotyping results. In contrast to current methods, the kit eliminates the need for optimization of PCR parameters such as Mg2+concentration, primer concentrations, and cycling conditions.|
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")。

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
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。

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

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