ULTRARAPID DNA SEQUENCING BY SURFACE PLASMON RESONANCE

尽管新一代基于机器人的DNA测序仪器即将发展，但用于破译人类基因组的技术——即荧光标记寡核苷酸的电泳分离——已经过时了，目前尚不清楚这项任务能否在2005年之前使用这些方法完成。因此，提出了一种全新的DNA测序方法，既不需要荧光标记的核苷酸也不需要分离。基于表面等离子体共振（SPR），一种已经被生化界广泛接受和使用的技术，其概念是在复制过程中监测胶体Au标记的DNA聚合酶与DNA衍生的SPR芯片表面的距离。互补核苷酸的结合导致胶体Ad:DNA聚合酶复合物沿着单链DNA移动，然后检测到反射率的变化。这项新技术的科学基础是胶体金纳米颗粒的接近对金薄膜反射率的巨大影响，PI已经通过开发基于SPR的超灵敏胶体金扩增免疫测定法利用了这一效应。如果本文提出的工作完全成功，即如果超快速DNA测序的概念在单通道和多通道SPR仪器中得到证明，那么整个人类基因组的测序可以在一年多的时间内完成。

Notwithstanding the impending development of a new generation of robotics-based instrumentation for DNA sequencing, the technology applied to deciphering the human genome-namely electrophoretic separation of fluorescently-tagged oligonucleotides-is old, and it is unclear whether the task can be accomplished by the 2005 using these methods. Accordingly, a radically new approach to DNA sequencing requiring neither fluorescently-labeled nucleotides nor separation is proposed. Based on surface plasmon resonance (SPR), a technique that has already gained acceptance and widespread use by the biochemical community, the concept is to monitor the distance of a colloidal Au- tagged DNA polymerase from the from the surface of a DNA-derivatized SPR chip during replication. Incorporation of complementary nucleotides leads to movement of the colloidal Ad:DNA polymerase complex along single-stranded DNA, which is then detected as a change in reflectivity. The scientific basis for this new technology is the extraordinarily large effect on Au thin film reflectivity caused by proximity of colloidal Au nanoparticles, an effect that has already been exploited by the PI through development of ultrasensitive colloidal Au-amplified immunoassays based on SPR. If the work proposed herein is fully successful, i.e. if the concept of ultrarapid DNA sequencing is demonstrated both in single-channel and multi-channel SPR instruments, the entire human genome can be sequenced in just over one year.