柏业贸易

概述

通过应用PyroHotStart技术，达到非特异性扩增最小化，PCR反应效率最大化从而有效扩增微量模板DNA。PyroHotStart技术是Bioneer独有的一项专利技术，它使用焦磷酸盐和焦磷酸酶通过减少PCR反应之前（零循环）发生的非特异性反应来提高特异性。另外，它通过除去在每个扩增循环中发生并用作PCR抑制剂的PPi来提高反应效率。

(a)在Zero cycle抑制PCR反应
制备PCR混合液后，使用PPi防止在达到预变性温度之前形成非特异性反应物。为了使DNA聚合酶具有活性，需要镁离子，因为添加到混合液中的PPi与镁离子具有高亲和力，可以通过保留镁离子来抑制PCR反应

(b) 变性（Denaturation）
在变形步骤中，当反应温度升高时，添加到混合液中的ppase会被激活，从而将PPi分解为Pi解离镁离子。解离的镁离子与DNA聚合酶结合并激活该酶催化PCR反应

(c)退火（Annealing） & 延伸（Extension）
随着PCR循环的进行，活化的DNA聚合酶会扩增目标DNA

特点与优势

兼容性
其出色的兼容性适用于多款的实时荧光定量PCR仪
便利性
每个PCR管均包含DNA聚合酶和PCR反应所需的所有必需组分，因此只需加入模板DNA，目标基因的探针和引物以及D.W即可进行PCR反应
稳定性
PCR反应混合液中包含稳定剂，以实现室温下运送，并比溶液型产品更稳定
再现性
ISO 9001质量体系下的批量生产可最大程度地减少批次之间的差异，即使在重复实验中也可得到可重复的实验结果

应用

• Real-Time Quantification of DNA/cDNA targets.

• Quantification of gene expression

• Microbial/Viral Pathogen Detection.

实验数据

Specificity activity

Figure. Real-Time PCR Data of AccuPower® Greenstar™ qPCR PreMix

AccuPower® Greenstar™ qPCR PreMix provides at least 7 orders of magnitude in dynamic range (10 fg ~ 10 ng/reaction). All data were obtained using Exicycler™ 96 Real-time Quantitative Thermal Block.

(A) : Amplification curve. Lambda DNA primers were added into AccuPower® Greenstar™ qPCR PreMix. A series of Lambda DNA positive control diluents were tested.
(B) : Standard curve. Data were obtained using Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer Co.).
(C) : Melting curve analysis of AccuPower® Greenstar™ qPCR PreMix. - The melting curve shows that only single amplified PCR product was obtained in all template range.

▶ Specifications

▶ Experimental data

Figure 1. Comparison of amplification quality between AccuPower® Greenstar™ qPCR PreMix and other supplier's master mixture

Amplification & Standard curve of AccuPower® Greenstar™ qPCR PreMix and other supplier's master mixture. Lambda DNA primers were added into AccuPower® Greenstar™ qPCR PreMix and other supplier's master mixture. A series of Lambda DNA positive control diluents were tested. Reaction mixtures were prepared and qPCR was performed according to each supplier's protocol. All data were obtained using ABI 7500 Fast Real-Time PCR machine (Applied Biosystems co.).

Stability

Figure 2. Stability test of AccuPower® Greenstar™ qPCR PreMix at 50℃ based on Accelerated Life test

Amplification curve of AccuPower® Greenstar™ qPCR PreMix after storage at 50℃ temperature. The amplification curve shows same Ct value when used template of same concentration in all data. All data were obtained using Exicycler™ 96 Real-time Quantitative Thermal Block.

A : Real-time PCR immediately after lyophilized (positive control)
B : Real-time PCR at 1 day
C : Real-time PCR at 3 days
D : Real-time PCR at 5 days
E : Real-time PCR at 6 days