Yguerabide J, Ceballos A
Department of Biology, University of California at San Diego, La Jolla 92093, USA.
Anal Biochem. 1995 Jul 1;228(2):208-20. doi: 10.1006/abio.1995.1341.
We present a quantitative fluorescence method for continuous measurement of DNA or RNA hybridization (including renaturation) kinetics using a fluorescent DNA intercalator. The method has high sensitivity and can be used with reaction volumes as small as 1 microliter and amounts of DNA around 1 ng. The method is based on the observations that (i) for the usual hybridization conditions, intercalators such as ethidium bromide bind (intercalate) to double-stranded DNA (dsDNA) but not single-stranded DNA or RNA and (ii) there is a large increase in fluorescence intensity when intercalators such as ethidium bromide bind to dsDNA. In this application, the intercalator can be considered as a quantitative indicator of dsDNA concentration. When a small amount of intercalator is added to a hybridizing solution, the fluorescence intensity of the intercalators increases with increase in dsDNA. The hybridization reaction can thus be monitored by continuously recording fluorescence intensity vs time. Because the amount of intercalator bound to dsDNA is not necessarily proportional to dsDNA concentration, the time-dependent fluorescence intensity graph is not identical to the kinetic graph [dsDNA] vs t. However, the fluorescence intensity vs time graph can easily be converted to the true [dsDNA] vs t graph by means of an experimental calibration graph of fluorescence intensity vs [dsDNA]. This calibration graph is obtained in a separate experiment using samples containing known amounts of dsDNA in the ethidium bromide buffer used in the kinetic measurement. We present results of experimental tests of the intercalator technique using ethidium bromide as an intercalator and DNA from Escherichia coli and lambda-phage and Poly(I)-Poly(C) RNA hybrids. These DNA and RNA samples have Cot1/2 values that cover a range of 10(6). Our experimental results show that (i) the kinetics of hybridization are not significantly perturbed by the intercalator at concentrations where no more than 10% of the binding sites on DNA or RNA hybrids are occupied, (ii) the kinetic graphs obtained by the intercalator fluorescence method and corrected with the calibration graph agree with kinetic graphs obtained by optical absorbance measurements at 260 nm, and (iii) the intercalator technique can be used in the different salt environments often used to increase the velocity of the hybridization reaction and at the hybridization temperatures (35-75 degrees C) normally used to minimize nonspecific hybridization.
我们提出了一种定量荧光方法，用于使用荧光DNA嵌入剂连续测量DNA或RNA杂交（包括复性）动力学。该方法具有高灵敏度，可用于小至1微升的反应体积和大约1纳克的DNA量。该方法基于以下观察结果：（i）在通常的杂交条件下，诸如溴化乙锭之类的嵌入剂会与双链DNA（dsDNA）结合（嵌入），但不会与单链DNA或RNA结合；（ii）当诸如溴化乙锭之类的嵌入剂与dsDNA结合时，荧光强度会大幅增加。在本应用中，嵌入剂可被视为dsDNA浓度的定量指标。当向杂交溶液中加入少量嵌入剂时，嵌入剂的荧光强度会随着dsDNA的增加而增加。因此，可以通过连续记录荧光强度随时间的变化来监测杂交反应。由于与dsDNA结合的嵌入剂的量不一定与dsDNA浓度成正比，所以荧光强度随时间变化的图与[dsDNA]随时间变化的动力学图并不相同。然而，通过荧光强度与[dsDNA]的实验校准图，可以轻松地将荧光强度随时间变化的图转换为真实的[dsDNA]随时间变化的图。该校准图是在一个单独的实验中获得的，使用的样品中含有在动力学测量中所用的溴化乙锭缓冲液中已知量的dsDNA。我们展示了使用溴化乙锭作为嵌入剂以及来自大肠杆菌和λ噬菌体的DNA和聚肌苷酸-聚胞苷酸（Poly(I)-Poly(C)）RNA杂交体的嵌入剂技术的实验测试结果。这些DNA和RNA样品的Cot1/2值覆盖了10^6的范围。我们的实验结果表明：（i）在DNA或RNA杂交体上不超过10%的结合位点被占据的浓度下，嵌入剂对杂交动力学没有显著干扰；（ii）通过嵌入剂荧光方法获得并经校准图校正的动力学图与通过260nm光吸收测量获得的动力学图一致；（iii）嵌入剂技术可用于通常用于提高杂交反应速度的不同盐环境以及通常用于最小化非特异性杂交的杂交温度（35 - 75摄氏度）。
