ULTRARAPID DNA SEQUENCING BY SURFACE PLASMON RESONANCE

尽管即将开发新一代以机器人为基础的DNA测序仪器，但应用于破译人类基因组的技术--即荧光标记的寡核苷酸的电泳分离--是旧的，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。基于表面等离子共振(SPR)，这项技术已经被生化界接受并广泛使用，其概念是在复制过程中监控胶体Au标记的DNA聚合酶与DNA衍生的SPR芯片表面的距离。互补核苷酸的掺入导致胶体Ad：DNA聚合酶复合体沿单链DNA移动，然后被检测为反射率的变化。这项新技术的科学基础是胶体Au纳米颗粒的接近对Au薄膜反射率造成的极大影响，PI已经通过开发基于SPR的超灵敏胶体Au放大免疫分析来利用这一影响。如果本文提出的工作完全成功，即如果在单通道和多通道SPR仪器上都证明了超快DNA测序的概念，那么整个人类基因组可以在一年多一点的时间内完成测序。

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.