Crane Bryan L, Hogan N Catherine, Sudo Hiroko, Thilly William G, Hunter Ian W
BioInstrumentation Laboratory, Department of Mechanical Engineering, and Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Anal Chem. 2005 Aug 15;77(16):5129-34. doi: 10.1021/ac050323j.
We have developed an instrument for monitoring real-time PCR using fluorescence anisotropy, enabling an assay chemistry in which the fluorescence from a labeled primer elucidates amplification. The instrument holds the sample temperature constant to within +/-0.03 degrees C during measurement in the extension phase of each PCR cycle and achieves 0.116 mP FA resolution. Primer conjugation with Alexa-Fluor 488, when compared with other fluorophores, is shown to provide the greatest FA range between primer and product. Comparable reproducibility and linearity of the crossing point for a range of target copy numbers is observed between the FA-based assay run in our instrument and the SYBR green assay run in commercial instrumentation. Reproducibility is also consistent with Poisson-distributed experimental error in aliquoting starting copies, a theoretical limit to instrument/assay performance.
我们开发了一种利用荧光各向异性监测实时PCR的仪器，实现了一种检测化学方法，其中标记引物发出的荧光可阐明扩增情况。该仪器在每个PCR循环的延伸阶段测量过程中，能将样品温度保持在±0.03摄氏度以内，并实现了0.116 mP FA分辨率。与其他荧光团相比，与Alexa-Fluor 488偶联的引物在引物与产物之间显示出最大的FA范围。在我们仪器上运行的基于FA的检测方法与在商业仪器上运行的SYBR green检测方法之间，观察到一系列目标拷贝数的交叉点具有相当的重现性和线性。重现性也与等分起始拷贝数时泊松分布的实验误差一致，这是仪器/检测性能的理论极限。
