Life Technologies Corporation

Guobin Luo

Theo Nikiforov

Peter B Vander Horn

Michael Previte

Cheng-Yao Chen

Daniel Mazur

Eileen Tozer

Barnett B Rosenblum

Marian Peris

Hongye Sun

Xinzhan Peng

Michael Phelan

Yufang Wang

Zhaohui Zhou

Sihong Chen

Hongyi Wang

Stefanie Yukiko Nishimura

Jamshid Temirov

Joseph M Beechem

Vi-En Choong

Mindy Landes

Kevin Heinemann

Joshua Shirley

David Light

Tommie Lloyd Lincecum

Ryan Jones

Marina Sedova

Mohammad Alanjary

Mark R Andersen

John F Davidson

Tommie Lincecum

The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, recombinant polymerases and biologically active fragments thereof are provided that allow for nucleic acid amplification. In some aspects, the disclosure provides recombinant polymerases that yield lower systematic error rates and/or improved accuracy, when used in sequencing by synthesis reactions as compared to a control polymerase. In one aspect, the disclosure relates to recombinant polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In another aspect, the recombinant polymerases are useful for the amplification of nucleic acid templates during PCR, emPCR, isothermal amplification, recombinase polymerase amplification, rolling circle amplification, strand displacement amplification and proximity ligation amplification. In some aspects, the disclosure relates to recombinant polymerases useful for the generation of nucleic acid libraries and/or nucleic acid templates.

Polymerase compositions and kits, and methods of using and making the same

The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, modified polymerases and biologically active fragment thereof are provided that allow for nucleic acid amplification. In one aspect, the disclosure relates to modified polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In some aspects, the disclosure relates to modified polymerases useful for the generation of nucleic acid libraries or nucleic acid templates for use in various downstream processes. In some aspects, the disclosure relates to the identification of homologous amino acid mutations that can be transferred across classes or families of polymerases to provide novel polymerases with altered catalytic properties. In some aspects, the disclosure provides modified polymerases having enhanced catalytic properties as compared to a reference polymerase.

Polymerase compositions, methods of making and using same

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation.

Nucleotide transient binding for sequencing methods

Provided are methods for preparing a library of target nucleic acid sequences, as well as compositions and uses therefor. Methods comprise contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; repairing the digested target amplicons; and amplifying the repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the plurality of adaptor compositions comprise a handle and a targeted nucleic acid sequence and optionally one or more tag sequences. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences. Resulting library compositions are useful for a variety of applications, including sequencing applications.

Library preparation methods and compositions and uses therefor

A method for depositing particles on a surface includes receiving a plurality of particles, a particle of the plurality of particles having a polymer matrix conjugated to nucleic acid strands, exposing the plurality of particles to a solution; and applying the plurality of particles to a surface following exposing the plurality of particles to the solution, particles of the plurality of particles depositing on the surface. The solution includes a magnesium salt in a range of 30 mM to 500 mM, a potassium salt in a range of 0.8 M to 1.0 M, a buffering agent in a range of 150 mM to 500 mM, and a surfactant in a range of 0.05% to 0.5%.

Device preparation using condensed nucleic acid particles

Provided herein are systems and methods for nucleotide incorporation reactions. The systems comprise polymerases having altered nucleotide incorporation kinetics and are linked to an energy transfer donor moiety, and nucleotide molecules linked with at least one energy transfer acceptor moiety. The donor and acceptor moieties undergo energy transfer when the polymerase and nucleotide are proximal to each other during nucleotide binding and/or nucleotide incorporation. As the donor and acceptor moieties undergo energy transfer, they generate an energy transfer signal which can be associated with nucleotide binding or incorporation. Detecting a time sequence of the generated signals, or the change in the signals, can be used to determine the order of the incorporated nucleotides, and can therefore be used to deduce the sequence of the target molecule.

Methods and apparatus for single molecule sequencing using energy transfer detection

A method of sequencing a nucleic acid strand includes receiving particles having nucleic acid strands coupled to a polymer matrix, exposing the particles to a solution including a condensing agent, and applying the particles to a surface, the particles depositing on the surface.

In some embodiments, the disclosure relates generally to methods as well as related compositions, systems, kits and apparatus comprising linking proteins to target compounds and/or to locations of interest using tethers. For example, the tether can be used to link the protein to a target compound, for example, to link an enzyme to a substrate. Similarly, the tether can be used to link the protein at or near a desired location on a surface. In one group of embodiments, the tether includes a polynucleotide and the target compound or location on the surface includes another polynucleotide that is capable of hybridizing to the tether. In such embodiments, the tether can be used to link the protein to the target compound or location using nucleic acid hybridization.

Linking methods, compositions, systems, kits and apparatuses

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Oceanside, CA, United States of America

Guobin Luo