• 对生殖细胞和体细胞CNV进行可靠、准确的相对定量
• 准确分辨细微的拷贝数差别
• 含有通用的多拷贝定量标准
• 优化的试剂、对照、以及实验方案可避免假阳性或假阴性结果
• 已使用TaqMan Copy Number Assays在多重qPCR实验中进行功能验证*

Type-it CNV Probe PCR +qC Kit利用基于TaqMan探针技术的多重qPCR技术，为人类生殖细胞和体细胞基因组拷贝数变异的相对定量提供了优化的解决方案。该试剂盒中含有创新的 预混液，可确保PCR反应效率高、特异性强。此外，该试剂盒还含有通用对照，可对CNV进行可靠验证。对照试剂覆盖了多拷贝位点，因此无需对每个基因 （GOI）的单拷贝对照进行验证。优化的定量PCR试剂和定量对照、以及专用实验方案（适用于自设计和商用TaqMan Copy Number Assays），可确保高度灵敏、可靠、准确的拷贝数定量检测结果。

原理

CNVs are known to affect approximately 10–20% of the human genome and have been associated with many types of cancer and various complex diseases, such as autism and autoimmune disorders. Quantitative PCR, with its ease of use, sensitivity, and scalability, is often the method of choice for CNV validation and association studies. A reference gene, whose copy number is presumed to be constant among the samples being compared, must first be defined. The copy number change of the genes of interest is then calculated based on the C_T difference between the GOI and reference gene in different samples (ΔΔC_T method for relative quantification). The Type-it CNV Probe PCR +qC Kit provides accurate relative quantification of both germline and somatic CNVs using TaqMan probe-based multiplex qPCR. The Type-it CNV Probe PCR +qC Kit includes a highly specific master mix and a patent-pending multi-copy quantification control reference assay for reliable evaluation of copy number changes of different GOI in various samples. Experience fast, convenient, and reliable CNV screening using any real-time cycler(see figure Successful screening of 9 different Coriell DNA samples and table “Features of the Type-it CNV Probe PCR +qC Kit”).
 

Highly specific master mix

The 2x Type-it Probe PCR Master Mix contains HotStarTaq Plus DNA Polymerase for a stringent hot start and highly specific and sensitive amplification, as well as an innovative PCR buffer and dNTPs. The master mix also contains ROX dye, and is suited for use with various real-time cyclers (see table “Suitable real-time instruments”). The 2x Type-it Probe PCR Master Mix is based on QIAGEN’s patented multiplex PCR chemistry and promotes specific primer annealing by stabilizing specifically bound primers, ensuring highly successful duplex PCR results. The ready-to-use master mix is specifically optimized for precise quantification of copy number variations by parallel amplification of the provided Type-it CNV Reference Probe Assay together with the genes of interest. The master mix ensures that the target sequence is amplified with the same efficiency and sensitivity in the duplex or multiplex PCR reaction as in a corresponding singleplex reaction. The Type-it CNV Probe PCR Buffer is based on QIAGEN’s dual-cation buffer system and ensures high specificity and efficiency during PCR. View this animation and discover the benefits of QIAGEN’s unique dual-cation PCR buffer. The ready-to-use master mix eliminates the need for optimization of PCR parameters, ensuring superior PCR performance at the first attempt (see figure Type-it CNV Probe PCR +qC Kit outperforms kit from Supplier L). The master mix also includes QIAGEN’s Q-Bond technology for fast cycling.
 

Universal multi-copy reference assay

The Type-it CNV Reference Probe Assay is a TaqMan probe-based assay and targets a multi-copy gene element in the human genome. It allows simultaneous amplification of the Type-it CNV Reference and user-defined GOI by multiplex, real-time PCR using sequence-specific probes for reliable quantification of CNVs. The Type-it CNV Reference Probe Assay is based on a multi-copy genetic element that is distributed on different chromosomes in the human genome. The Type-it CNV Reference Assay is consistent among diverse human populations, assuring successful and universal application (see figure Highly reliable multi-copy quantification control assay). Compared with typical single-copy reference genes, this patent-pending multi-copy reference assay ensures superior reliability in CNV quantification, and eliminates the need for optimization and validation of reference assays for each GOI under investigation.

操作流程

The Type-it CNV Probe PCR +qC Kit is developed for use on all real-time cyclers, including the Rotor-Gene Q, and instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent (see table “Suitable real-time instruments”). Using the optimized protocols provided with the kit, duplex or multiplex PCR is carried out in the presence of ROX passive reference dye. Both somatic and germline CNVs can be successfully investigated by simply setting up reactions with assays for the GOI and following the preoptimized protocols.

Suitable real-time instruments

Supplier	Thermal cycler
QIAGEN	Rotor-Gene Q
Life Technologies	Applied Biosystems 7500 (all series), Applied Biosystems 7300 and 7300, ABI PRISM 7000 and 7700, Applied Biosystems StepOne and StepOnePlus, ViiA 7
Stratagene	Mx300P and Mx3005P
Bio-Rad	iCycler iQ, CFX
Roche	LightCycler 480, LightCycler 2
Eppendorf	RealPlex

Fast and straightforward procedure for precise results

The unique components of the Type-it CNV Probe PCR +qC Kit enable fast and reliable CNV validation and screening (see table “Features of the Type-it CNV Probe PCR +qC Kit”). PCR run times are reduced by up to 40% through patented Q-Bond technology and stringent HotStarTaq Plus DNA Polymerase. The streamlined protocol omits the need for time-consuming optimization of critical factors such as template amount and cycling conditions on different cyclers. Use of the patent-pending universal quantification control reference assay eliminates the need for validation of individual single-copy elements. Calculation of copy number alterations is therefore accomplished with greater ease and accuracy, while the risk of false-positives/negatives is reduced, resulting in time and cost savings.

Features of the Type-it CNV Pobe PCR +qC Kit

Kit component	Features
2x Type-it Probe PCR Master Mix	Accurate relative quantification of CNVs Suitable for use with various cyclers Multiplex PCR master mix validated with TaqMan Copy Number Assays
HotStarTaq Plus DNA Polymerase (included in the master mix)	Setup at room temperature Highly specific and sensitive PCR
Type-it CNV Probe PCR Buffer (included in the master mix)	Increased specificity and efficiency Balanced NH4+ and K+ concentrations Fast-cycling feature with Q-Bond technology Simultaneous quantification of up to three GOIs
Type-it CNV Reference Probe Assay	Multi-copy element serves as universal quantification control Optimization of reference for each GOI not required

应用

All somatic and germline CNVs in the human genome can be reliably quantified using the Type-it CNV Probe PCR +qC Kit in various applications, including cancer research, and in the study of various complex diseases, such as autism and autoimmune disorders.