Reading Single DNA with DNA Polymerase Followed by Atomic Force Microscopy

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 atomi...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2014-10, Vol.136 (39), p.13754-13760
Main Authors: Kim, Youngkyu, Kim, Eung-Sam, Lee, Yoonhee, Kim, Joung-Hun, Shim, Bong Chu, Cho, Seong Moon, Lee, Jeong Soo, Park, Joon Won
Format: Article
Language:English
Subjects:
Base Sequence
DNA - chemistry
DNA - metabolism
DNA - ultrastructure
DNA-Directed DNA Polymerase - metabolism
DNA-Directed DNA Polymerase - ultrastructure
Microscopy, Atomic Force
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Summary: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’s 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.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja5063983