Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides
Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides
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DOI：
10.1016/s0006-3495(96)79473-0
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发表时间：
1996-11-01
影响因子：
3.4
通讯作者：
Mirzabekov, AD
中科院分区：
文献类型：
--
作者：
Livshits, MA;Mirzabekov, AD
一种新的DNA测序方法是利用含有一组固定寡核苷酸的微芯片进行杂交。本文对脱氧核苷酸分子在聚丙烯酰胺凝胶层中的杂交动力学进行了理论分析。分析表明，杂交细胞中结合的DNA的空间分布和数量的长期进化是由“延迟扩散”控制的，即DNA的扩散被与固定的寡核苷酸分子的反复结合和解离所中断。延迟扩散决定了在细胞中建立最终平衡状态的特征时间，即结合DNA数量最大且分布均匀的状态。在具有最稳定、最完美的双链的细胞中，延迟扩散的特征时间（与平衡结合常数和结合位点的浓度成正比）可能比实际杂交过程的持续时间更长。观察到完全匹配杂交细胞中标记DNA的荧光不均匀（边缘较亮），间接证实了这一结论。为了最优地区分完全双工和不匹配双工，在所有细胞的低温非饱和条件下，杂交过程应达到平衡。凝胶中完美和非完美双链之间的动力学差异允许通过杂交后在低温下额外洗涤进一步改善识别。
A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by ''retarded diffusion,'' i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic lime of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges), For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells, The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.