Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase.
Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase.
复制标题
通过监测 DNA 聚合酶的酶活性进行定量聚合酶链反应。
DOI：
10.1006/abio.1993.1014
复制
发表时间：
1993
影响因子：
2.9
通讯作者：
Viscidi,R
中科院分区：
文献类型：
--
作者：
Yang,B;Yolken,R;Viscidi,R
通过聚合酶链反应（PCR）的核酸扩增在灵敏度方面是非常强大的技术，但在进行精确定量的能力方面是有限的。虽然在输入靶序列的拷贝数和PCR产物的拷贝数之间存在理论上的相关性，但是这种关系的定量性质被反应条件中不可预测的变化以及可能存在于样品制备物中的抑制性和/或刺激性物质所掩盖，特别是那些来自生物液体的物质。为了可靠地估计来自PCR产物的输入DNA靶的拷贝数，我们设计了与DNA/RNA杂交和酶促免疫检测技术相结合的内部和外部控制系统。内部对照系统用于监测扩增效率并校正抑制剂或刺激物对DNA扩增效率的影响。该测定是定量的，非同位素的，并可广泛应用于评估的DNA的量存在于各种各样的准备。
Nucleic acid amplification by polymerase chain reaction (PCR) is a very powerful technique in terms of sensitivity but is limited in terms of ability to perform accurate quantitation. While there is a theoretical correlation between copies of input target sequence and those of PCR product, the quantitative nature of this relationship is obscured by unpredictable variations in reaction conditions and by inhibitory and/or stimulatory substances which might be present in sample preparations, especially those derived from biological fluids. To reliably estimate copies of input DNA target from PCR product, we designed a combination of internal and external control systems coupled to DNA/RNA hybridization and enzymatic immunodetection techniques. The internal control system served to monitor amplification efficiency and to correct for the effects of inhibitors or stimuli on the efficiency of the DNA amplification. The assay is quantitative, nonisotopic, and can be widely applied to assessment of the quantity of DNA present in a wide range of preparations.