Reading Single DNA with DNA Polymerase Followed by Atomic Force Microscopy

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• 作者：Youngkyu Kim ; Eung-Sam Kim ; Yoonhee Lee ; Joung-Hun Kim ; Bong Chu Shim ; Seong Moon Cho ; Jeong Soo Lee ; Joon Won Park
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：October 1, 2014
• 年：2014
• 卷：136
• 期：39
• 页码：13754-13760
• 全文大小：376K
• ISSN：1520-5126

文摘

The importance of DNA sequencing in the life sciences and personalized medicine is continually increasing. Single-molecule sequencing methods have been developed to analyze DNA directly without the need for amplification. Here, we present a new approach to sequencing single DNA molecules using atomic force microscopy (AFM). In our approach, four surface-conjugated nucleotides were examined sequentially with a DNA polymerase-immobilized AFM tip. By observing the specific rupture events upon examination of a matching nucleotide, we could determine the template base bound in the polymerase鈥檚 active site. The subsequent incorporation of the complementary base in solution enabled the next base to be read. Additionally, we observed that the DNA polymerase could incorporate the surface-conjugated dGTP when the applied force was controlled by employing the force-clamp mode.