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'-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.
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027; Genia Technologies, Santa Clara, CA 95050;
Carl W
CW
Fuller
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Shiv
S
Kumar
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Mintu
M
Porel
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Minchen
M
Chien
Genia Technologies, Santa Clara, CA 95050;
Arek
A
Bibillo
Department of Genetics, Harvard Medical School, Boston, MA 02115;
P Benjamin
PB
Stranges
Genia Technologies, Santa Clara, CA 95050;
Michael
M
Dorwart
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Chuanjuan
C
Tao
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Zengmin
Z
Li
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Wenjing
W
Guo
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Shundi
S
Shi
Genia Technologies, Santa Clara, CA 95050;
Daniel
D
Korenblum
Genia Technologies, Santa Clara, CA 95050;
Andrew
A
Trans
Genia Technologies, Santa Clara, CA 95050;
Anne
A
Aguirre
Genia Technologies, Santa Clara, CA 95050;
Edward
E
Liu
Genia Technologies, Santa Clara, CA 95050;
Eric T
ET
Harada
Genia Technologies, Santa Clara, CA 95050;
James
J
Pollard
Genia Technologies, Santa Clara, CA 95050;
Ashwini
A
Bhat
Genia Technologies, Santa Clara, CA 95050;
Cynthia
C
Cech
Genia Technologies, Santa Clara, CA 95050;
Alexander
A
Yang
Genia Technologies, Santa Clara, CA 95050;
Cleoma
C
Arnold
Department of Genetics, Harvard Medical School, Boston, MA 02115;
Mirkó
M
Palla
Genia Technologies, Santa Clara, CA 95050;
Jennifer
J
Hovis
Genia Technologies, Santa Clara, CA 95050;
Roger
R
Chen
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Irina
I
Morozova
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
Sergey
S
Kalachikov
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027;
James J
JJ
Russo
Semiconductor and Dimension Metrology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899;
John J
JJ
Kasianowicz
Genia Technologies, Santa Clara, CA 95050;
Randy
R
Davis
Genia Technologies, Santa Clara, CA 95050;
Stefan
S
Roever
Department of Genetics, Harvard Medical School, Boston, MA 02115; gchurch@genetics.med.harvard.edu dj222@columbia.edu.
George M
GM
Church
Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; Department of Chemical Engineering, Columbia University, New York, NY 10027; Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, NY 10032 gchurch@genetics.med.harvard.edu dj222@columbia.edu.
Jingyue
J
Ju
D001483:Base Sequence; D003199:Computer Systems; D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D058608:Nanopores / Q000648:ultrastructure*; D009711:Nucleotides / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D011108:Polymers / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D009711:Nucleotides; D011108:Polymers; D004247:DNA
DNA sequencing by synthesis; chip array; nanopore; polymer-tagged nucleotides; single-molecule sequencing