Lee Hye Jin, Li Yuan, Wark Alastair W, Corn Robert M
Department of Chemistry, University of California-Irvine, Irvine, CA 92697, USA.
Anal Chem. 2005 Aug 15;77(16):5096-100. doi: 10.1021/ac050815w.
This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.
本文描述了一种新方法，该方法利用核酸外切酶III与3'端终止的DNA微阵列相结合，通过表面等离子体共振（SPR）成像对单链DNA（ssDNA）进行扩增检测。当将核酸外切酶III和靶DNA同时引入到3'端终止的ssDNA微阵列中时，靶单链DNA的杂交吸附会导致探针单链DNA链的核酸外切酶III方向依赖性水解，并将完整的靶单链DNA释放回溶液中。靶单链DNA重新吸附到另一个探针上会产生一个重复的水解过程，随着时间的推移，SPR成像信号会出现显著的负变化。文中展示的实验证明了核酸外切酶III与双链DNA的方向依赖性表面酶反应，以及这种新的酶促扩增SPR成像过程，其对16聚体靶单链DNA的检测限为10 - 100 pM。这比之前仅基于杂交吸附而无酶促扩增的SPR成像检测单链DNA的测量结果提高了10² - 10³倍。
