Open AccessReading Single DNA with DNA Polymerase Followed by Atomic Force Microscopy
Author(s) -
Youngkyu Kim,
EungSam Kim,
Yoon-Hee Lee,
JoungHun Kim,
Bong Chu Shim,
Seong Moon Cho,
Jeong Soo Lee,
Chong-Yun Park
Publication year - 2014
Publication title -
journal of the american chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.115
H-Index - 612
eISSN - 1520-5126
pISSN - 0002-7863
DOI - 10.1021/ja5063983
Subject(s) - chemistry , dna polymerase , dna clamp , base pair , atomic force microscopy , polymerase , single molecule real time sequencing , dna , biophysics , nucleotide , nanotechnology , polymerase chain reaction , dna sequencing , computational biology , microbiology and biotechnology , biochemistry , gene , biology , reverse transcriptase , dna sequencer , materials science
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.