Techniques for single molecule sequencing

Bioimaging Pub Date : 2001-05-11 DOI:10.1002/1361-6374(199709)5:3<139::AID-BIO8>3.0.CO;2-%23

Klaus Dörre, Susanne Brakmann, Michael Brinkmeier, Kyung-Tae Han, Katja Riebeseel, Petra Schwille, Jens Stephan, Timm Wetzel, Markus Lapczyna, Michael Stuke, Raoul Bader, Michael Hinz, Hartmut Seliger, Johan Holm, Manfred Eigen, Rudolf Rigler

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Abstract

A method is described that demonstrates a new technique for rapid and high-throughput single molecule sequencing. This sequencing technique is based on the successive enzymatic degradation of fluorescently labeled single DNA molecules, and the detection and identification of the released monomer molecules according to their sequential order in a microstructured channel.

The detection technique is evolved from confocal fluorescence microscopy, with two different laser sources to excite the individual mononucleotides that are either labeled with tetramethylrhodamine (TMR) or Cyanine5 (Cy5). The handling of DNA which is immobilized on carrier beads, and the detection of the cleaved monomers is performed in optically transparent and biochemically inert microstructures (glass or PMMA) with detection channels of 7 μ × 10 μm.

The projected rate of sequencing is ≈100 bases min⁻¹, dependent solely on the rate of the enzymatic DNA cleavage.

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单分子测序技术

介绍了一种快速、高通量单分子测序的新方法。这种测序技术是基于连续的酶降解荧光标记的单个DNA分子，并根据其在微结构通道中的顺序检测和鉴定释放的单体分子。检测技术是从共聚焦荧光显微镜发展而来的，用两种不同的激光源来激发用四甲基罗丹明(TMR)或氰胺5 (Cy5)标记的单个单核苷酸。固定在载体珠上的DNA的处理和裂解单体的检测是在光学透明和生物化学惰性的微结构(玻璃或PMMA)中进行的，检测通道为7 μ × 10 μm。预计测序率为≈100个碱基min - 1，仅取决于酶促DNA切割的速率。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Bioimaging

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