Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5�...

Celý popis

Uložené v:
Podrobná bibliografia
Vydané v:Proceedings of the National Academy of Sciences - PNAS Ročník 113; číslo 19; s. 5233
Hlavní autori: Fuller, Carl W, Kumar, Shiv, Porel, Mintu, Chien, Minchen, Bibillo, Arek, Stranges, P Benjamin, Dorwart, Michael, Tao, Chuanjuan, Li, Zengmin, Guo, Wenjing, Shi, Shundi, Korenblum, Daniel, Trans, Andrew, Aguirre, Anne, Liu, Edward, Harada, Eric T, Pollard, James, Bhat, Ashwini, Cech, Cynthia, Yang, Alexander, Arnold, Cleoma, Palla, Mirkó, Hovis, Jennifer, Chen, Roger, Morozova, Irina, Kalachikov, Sergey, Russo, James J, Kasianowicz, John J, Davis, Randy, Roever, Stefan, Church, George M, Ju, Jingyue
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States 10.05.2016
Predmet:
Base Sequence
Computer Systems
Conductometry - instrumentation
DNA - chemistry
DNA - genetics
Equipment Design
Equipment Failure Analysis
Nanopores - ultrastructure
Nucleotides - genetics
Oligonucleotide Array Sequence Analysis - instrumentation
Oligonucleotide Array Sequence Analysis - methods
Polymers - chemistry
Sequence Analysis, DNA - instrumentation
Sequence Analysis, DNA - methods
Staining and Labeling - methods
polymer-tagged nucleotides
DNA sequencing by synthesis
chip array
nanopore
single-molecule sequencing
ISSN:1091-6490, 1091-6490
On-line prístup:Zistit podrobnosti o prístupe
Tagy: Pridať tag
Žiadne tagy, Buďte prvý, kto otaguje tento záznam!
Popis
Shrnutí:DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.
Bibliografia:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:1091-6490
1091-6490
DOI:10.1073/pnas.1601782113