ULTRARAPID DNA SEQUENCING BY SURFACE PLASMON RESONANCE

通过表面等离子体共振进行超快速 DNA 测序

• 批准号: 6181830
• 负责人: CHRISTINE D KEATING
• 金额: $ 9.18万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-19 至 2001-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181830
• 关键词: DNA; DNA directed DNA polymerase; DNA footprinting; genetic mapping; genome; molecular dynamics; nanotechnology; nucleic acid sequence; surface plasmon resonance; technology /technique development

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.

尽管新一代基于机器人的DNA测序仪器即将开发，但用于破译人类基因组的技术-即荧光标记的磷脂的电泳分离-是旧的，并且不清楚是否可以在2005年之前完成任务。因此，提出了一种既不需要荧光标记的核苷酸也不需要分离的DNA测序的全新方法。基于表面等离子体共振（SPR），一种已经被生物化学界接受和广泛使用的技术，其概念是在复制期间监测胶体Au标记的DNA聚合酶与DNA衍生的SPR芯片表面的距离。互补核苷酸的掺入导致胶体Ad：DNA聚合酶复合物沿着单链DNA移动，然后将其检测为反射率的变化。这项新技术的科学基础是由于胶体Au纳米颗粒的接近而对Au薄膜反射率产生的非常大的影响，PI已经通过开发基于SPR的超灵敏胶体Au放大免疫测定来利用这种影响。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，则整个人类基因组可以在一年多的时间内测序。