DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants
DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants
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DOI：
10.1093/nar/27.7.1719
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发表时间：
1999-04-01
影响因子：
14.9
通讯作者：
Kelso, DM
中科院分区：
文献类型：
--
作者：
Stevens, PW;Henry, MR;Kelso, DM
许多DNA探针分析使用包被寡核苷酸的微粒从溶液中捕获互补核酸。在这些分析的开发过程中，以及在其他基于颗粒的核酸应用中，了解在各种实验条件下预期的双链生成量和捕获的寡核苷酸在颗粒表面的包被密度都是有用的。我们研究了DNA探针微粒分析的最简单形式：将粒子结合的捕获寡核苷酸与其溶液相补体杂交。将荧光素标记的溶液相寡核苷酸与不同数量的颗粒杂交，并测量平衡时标记的寡核苷酸留在溶液中的量。我们为非自互补序列的双分子杂交提出了一个简单的两态、全有或全无模型，该模型可以用来从杂交数据中计算平衡解离常数(Kd)。在Kd值和反应中捕获探针的浓度相对于反应中标记的互补寡核苷酸浓度较小的实验条件下，也可以确定捕获探针在粒子表面的密度。颗粒杂化的Kd值与溶液相热力学参数的Kd值不同。在较高温度下，在颗粒上杂交比在溶液中杂交更有效。
Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant (Kd) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.