WO2011040971A3 - Generation of modified polymerases for improved accuracy in single molecule sequencing - Google Patents

Generation of modified polymerases for improved accuracy in single molecule sequencing Download PDF

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Publication number
WO2011040971A3
WO2011040971A3 PCT/US2010/002659 US2010002659W WO2011040971A3 WO 2011040971 A3 WO2011040971 A3 WO 2011040971A3 US 2010002659 W US2010002659 W US 2010002659W WO 2011040971 A3 WO2011040971 A3 WO 2011040971A3
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WO
WIPO (PCT)
Prior art keywords
polymerases
dna
polymerase
single molecule
molecule sequencing
Prior art date
Application number
PCT/US2010/002659
Other languages
French (fr)
Other versions
WO2011040971A9 (en
WO2011040971A2 (en
Inventor
Sonya Clark
Arek Bibillo
Paul Peluso
Fred Christians
Molly He
Insil Park
Harold Lee
Keith Bjornson
Lei Jia
Robin Emig
Original Assignee
Pacific Biosciences Of California, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pacific Biosciences Of California, Inc. filed Critical Pacific Biosciences Of California, Inc.
Publication of WO2011040971A2 publication Critical patent/WO2011040971A2/en
Publication of WO2011040971A9 publication Critical patent/WO2011040971A9/en
Publication of WO2011040971A3 publication Critical patent/WO2011040971A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include reduced reaction rates at one or more steps of the polymerase kinetic cycle, increased closed polymerase/DNA complex stability, enhanced metal ion coordination, reduced exonuclease activity, decreased branching fractions, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.
PCT/US2010/002659 2009-09-30 2010-09-30 Generation of modified polymerases for improved accuracy in single molecule sequencing WO2011040971A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27804109P 2009-09-30 2009-09-30
US61/278,041 2009-09-30

Publications (3)

Publication Number Publication Date
WO2011040971A2 WO2011040971A2 (en) 2011-04-07
WO2011040971A9 WO2011040971A9 (en) 2011-08-04
WO2011040971A3 true WO2011040971A3 (en) 2011-11-03

Family

ID=43826830

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/002659 WO2011040971A2 (en) 2009-09-30 2010-09-30 Generation of modified polymerases for improved accuracy in single molecule sequencing

Country Status (1)

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WO (1) WO2011040971A2 (en)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9399766B2 (en) * 2012-10-01 2016-07-26 Pacific Biosciences Of California, Inc. Recombinant polymerases for incorporation of protein shield nucleotide analogs
WO2015061409A1 (en) * 2013-10-23 2015-04-30 University Of Washington Through Its Center For Commercialization Universal protein tag for double stranded nucleic acid delivery
CN105980580B (en) 2013-11-17 2020-03-03 宽腾矽公司 Optical system and assay chip for detecting, detecting and analyzing molecules
WO2016022998A2 (en) 2014-08-08 2016-02-11 Quantum-Si Incorporated Integrated device for temporal binning of received photons
EP3194934B1 (en) 2014-08-08 2024-03-06 Quantum-Si Incorporated Integrated device for use with an external light source for probing, detecting, and analyzing molecules by luminescence lifetime measurements
KR20220165282A (en) 2014-08-08 2022-12-14 퀀텀-에스아이 인코포레이티드 Optical system and assay chip for probing, detecting, and analyzing molecules
WO2017109262A1 (en) * 2015-12-24 2017-06-29 Consejo Superior De Investigaciones Científicas (Csic) Bacteriophage phi29 dna polymerase variants having improved thermoactivity
US11312944B2 (en) * 2016-12-19 2022-04-26 Quantum-Si Incorporated Polymerizing enzymes for sequencing reactions
CN108129571A (en) * 2017-12-25 2018-06-08 上海捷瑞生物工程有限公司 Taq DNA ligase fusion proteins
US20210355459A1 (en) * 2018-10-11 2021-11-18 Bgi Shenzhen Phi29 DNA polymerase mutant with improved thermal stability and use thereof in sequencing
EP3976771A1 (en) 2019-06-28 2022-04-06 Quantum-si Incorporated Polymerizing enzymes for sequencing reactions
CN113122517B (en) * 2021-03-24 2023-02-14 深圳清华大学研究院 Polymerase mutant and application thereof
CN113223607B (en) * 2021-05-28 2023-10-20 北京化工大学 Method for randomly generating heparin analogue structural coordinates in batch by adopting smiles algorithm

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008051530A2 (en) * 2006-10-23 2008-05-02 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008051530A2 (en) * 2006-10-23 2008-05-02 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARUNAS LAGUNAVICIUS ET AL.: "Duality of polynucleotide substrates for Phi29 DNA polymerase: 3' -5' RNase activity of the enzyme", RNA, vol. 14, no. 3, 2008, pages 503 - 513 *
BORJA IBARRA ET AL.: "Proofreading dynamics of a processive DNA polymerase", THE EMBO JOURNAL, vol. 28, no. 18, 2009, pages 2794 - 2802 *
DATABASE GENBANK 16 June 2009 (2009-06-16), "RecName: Full=DNA polymerase; AltName: Full=Early protein GP2", Database accession no. P03680 *
MARIA A. BLASCO ET AL.: "29 DNA Polymerase Active Site. The conserved amino acid motif "Kx3NSxYG" is involved in template-primer binding and dNTP selection", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 22, 1993, pages 16763 - 16770, XP002956405 *

Also Published As

Publication number Publication date
WO2011040971A9 (en) 2011-08-04
WO2011040971A2 (en) 2011-04-07

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