State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Talanta. 2010 Apr 15;81(1-2):418-23. doi: 10.1016/j.talanta.2009.12.018. Epub 2009 Dec 22.
Inherent problems exist with sequencing-by-synthesis (SBS) methods which use fluorescein-labeled nucleotide incorporation into a target template based on a polymerase chain reaction (PCR). These problems include lowering the cost of sequencing and the removal of fluorescence in DNA sequencing for further reading. How can these sequencing problems be resolved? We present a sequencing strategy which we call an extension-quenching-extension sequencing on a microarray based on a two-primer hyperbranched rolling circle amplification (HRCA). The microarray is a high throughput and low-cost tool. The template on a microarray for SBS was prepared by HRCA. The Cy 5-labeled deoxyribonucleoside triphosphate (dNTP) species were incorporated in the extension reactions. We discovered that copper (CuSO(4)) can quench the fluorescence in DNA sequencing because it exhibits an energy-transfer mechanism of quenching from the fluorescein to the bound Cu(2+) ion. The fluorescein label needs to be destroyed after the readout by CuSO(4) before further reading is possible. This paper describes the process used which discovered a successful combination of temperature, concentration, and duration of use for CuSO(4) which successfully quenched the fluorescence. In this experiment, we used a known sequence as a template in order to provide a strategy for sequencing.
基于聚合酶链反应（PCR）的荧光标记核苷酸掺入靶模板的测序合成（SBS）方法存在固有问题。这些问题包括降低测序成本和去除 DNA 测序中的荧光以便进一步读取。如何解决这些测序问题？我们提出了一种在微阵列上基于双引物超支化滚环扩增（HRCA）的扩展-猝灭-扩展测序策略。微阵列是一种高通量、低成本的工具。通过 HRCA 制备用于 SBS 的微阵列模板。在延伸反应中掺入 Cy5 标记的脱氧核苷三磷酸（dNTP）。我们发现铜（CuSO（4））可以猝灭 DNA 测序中的荧光，因为它表现出从荧光素到结合的 Cu（2+）离子的能量转移猝灭机制。在进一步读取之前，需要在 CuSO（4）读取后破坏荧光素标记。本文描述了所使用的过程，发现了成功的 CuSO（4）组合，包括温度、浓度和使用时间，可成功猝灭荧光。在这个实验中，我们使用了已知的序列作为模板，以提供测序的策略。
