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PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis

Published

Author(s)

Shiv Kumar, Tao Chuanjuan, Chien Minchen, Hellner Brittney, Joseph W. Robertson, Arvind Balijepalli, Li Zengmin, James J. Russo, Joseph E. Reiner, John J. Kasianowicz, Jingyue Ju

Abstract

There is a significant need to accurately sequence single DNA and RNA molecules for personalized medicine. We describe a novel nanopore-based sequencing by synthesis (SBS) strategy that will accurately differentiate at single molecule level four different sized tags that are initially attached to the 5'-phosphate of each nucleotide. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released by phosphodiester bond formation between the -phosphate of the tagged nucleotide and the 3'-OH group of the previous nucleotide. The released tags will enter a nanopore in the order they were released, and effect a unique ionic current blockade signature due to their size, shape and charge, thereby determining the DNA sequence electronically at single molecule level with single base resolution. As proof-of-principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient incorporation of these modified nucleotides during the polymerase reaction, and better than 6 s tag discrimination between the four tags based on the degree to which they reduce the nanopore ionic current. In the future, the molecular approach described here coupled with polymerase covalently attached to the nanopores in an array format, will yield a single-molecule nanopore-based SBS platform.
Citation
Scientific Reports

Keywords

DNA sequencing, nanopore, sequencing-by-synthesis

Citation

Kumar, S. , Chuanjuan, T. , Minchen, C. , Brittney, H. , Robertson, J. , Balijepalli, A. , Zengmin, L. , Russo, J. , Reiner, J. , Kasianowicz, J. and Ju, J. (2012), PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis, Scientific Reports, [online], https://doi.org/10.1038/srep00684, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=911143 (Accessed September 27, 2022)
Created September 20, 2012, Updated October 12, 2021