Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer
Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer
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DOI：
10.1006/abio.1999.4344
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发表时间：
1999-12-15
影响因子：
2.9
通讯作者：
Kelso, DM
中科院分区：
文献类型：
--
作者：
Henry, MR;Stevens, PW;Kelso, DM
当捕获的寡核苷酸拴在平面上时，质量输运限制会影响固相核酸杂交的动力学。然而，根据扩散理论，寡核苷酸在微粒上的杂交应该受到反应速率的限制。为了初步了解微粒杂交反应的动力学，我们开发了一种荧光共振能量转移方法来监测微粒上的寡核苷酸杂交。在表面密度为20%、40%和80%饱和度的情况下，用荧光化的低聚物包裹微颗粒，与四甲基罗丹明标记的互补寡核苷酸杂交，并随着时间的推移监测荧光素信号在颗粒表面发生杂交时的猝灭。比较了微粒杂交和液相杂交的结合速率常数。颗粒表面杂交的速率常数比溶液中杂交的速率常数小一个数量级，但将捕获寡核苷酸的表面密度降低到饱和的20%可提高颗粒的杂交速率。尽管双分子反应模型充分描述了溶液相杂交动力学，但微颗粒上的寡核苷酸杂交并不符合该模型，而是表现为双相反应动力学。基于两种不同的推理思路，我们认为基于微粒的寡核苷酸杂交在我们的系统中确实是反应速率有限的，而不是扩散速率有限的。(C) 1999学术出版社。
When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate Limited. (C) 1999 Academic Press.