Towery R B, Fawcett N C, Zhang P, Evans J A
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg 39406-5043, USA.
Biosens Bioelectron. 2001 Jan;16(1-2):1-8. doi: 10.1016/s0956-5663(00)00126-3.
Hybridization rates of sheared, genomic E. coli DNA in 0.14 M, pH 6.7 phosphate buffer at 65 degrees C were determined by: (1) observing the rate of absorbance decrease at 260 nm due to self-hybridization in solution; and (2) measurement of the rate of mass increase caused by hybridization between DNA in solution and DNA photografted to polystyrene. The latter measurement was done using a quartz crystal microbalance (QCM). In both the spectrophotometric and QCM experiments the probe was identical to the target, as both were taken from the same sample of sheared E. coli DNA. In the QCM measurements, viscoelastic effects were made negligible by drying the biopolymer layer on the QCM's surface before taking the frequency readings. Our purpose was to explore the effect of immobilizing DNA on its hybridization rate constant. A second-order constant of 2.32 +/- 0.09 x 10(-6) ml microg(-1) s(-1), n = 14, for hybridization in solution was obtained spectrophotometrically, while the QCM experiment gave a constant of 2.2 +/- 0.3 x 10(-6) ml microg(-1) s(-1), n = 6. These values are not statistically different. The reaction half-lives for the spectrophotometric and QCM experiments were 6.5 h and 13 min, respectively. The shorter half-life on the QCM can be explained solely by the much greater reactant concentration in the QCM experiment. About 25% of the DNA was inactivated by the attachment reaction. After correcting for this, the surface-attached DNA hybridized with the same rate constant as DNA free in solution. Therefore, it is concluded that, in these specific experiments with genomic DNA, the immobilized regions must have been short compared to the length of the molecules. The data demonstrate the high hybridization rate obtainable when nucleic acids are hybridized in a thin-film, micro-volume reaction on a non-porous surface.
在65摄氏度下，于0.14M、pH值为6.7的磷酸盐缓冲液中，通过以下方法测定剪切后的基因组大肠杆菌DNA的杂交速率：（1）观察由于溶液中自身杂交导致的260nm处吸光度下降速率；（2）测量溶液中的DNA与光接枝到聚苯乙烯上的DNA之间杂交引起的质量增加速率。后一种测量使用石英晶体微天平（QCM）进行。在分光光度法和QCM实验中，探针与靶标相同，因为两者均取自同一剪切后的大肠杆菌DNA样品。在QCM测量中，通过在读取频率之前干燥QCM表面的生物聚合物层，使粘弹性效应可忽略不计。我们的目的是探究固定化DNA对其杂交速率常数的影响。通过分光光度法获得溶液中杂交的二级常数为2.32±0.09×10⁻⁶mlμg⁻¹s⁻¹，n = 14，而QCM实验得到的常数为2.2±0.3×10⁻⁶mlμg⁻¹s⁻¹，n = 6。这些值在统计学上没有差异。分光光度法和QCM实验的反应半衰期分别为6.5小时和13分钟。QCM上较短的半衰期仅可由QCM实验中反应物浓度高得多来解释。约25%的DNA因附着反应而失活。校正此因素后，表面附着的DNA与溶液中游离DNA以相同的速率常数杂交。因此，可以得出结论，在这些基因组DNA的特定实验中，固定化区域与分子长度相比必定较短。数据表明，当核酸在无孔表面上进行薄膜微体积反应杂交时，可获得较高的杂交速率。
