Nucleotide transient binding for sequencing methods

Patent metadata

Field	Value
Publication number	US-2016208318-A1
Application number	US-201614991230-A
Country	US
Kind code	A1
Filing date	Jan 8, 2016
Priority date	Jun 5, 2009
Publication date	Jul 21, 2016
Grant date	—

Abstract

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.

First claim

1 - 20 . (canceled)

21 . A method for identifying a nucleotide bound to a polymerase, comprising:
transiently binding in a template-dependent manner, and without polymerizing, a first type of nucleotide to a first polymerase in the presence of a cation that inhibits nucleotide incorporation by the first polymerase,
wherein the first polymerase is included in a first immobilized complex comprising (i) the first polymerase, (ii) a first template nucleic acid molecule, and (iii) a first polymerization initiation site,
wherein the first immobilized complex is linked to a first site on a surface and the surface includes a plurality of immobilized complexes linked to other sites on the same surface
wherein the first polymerase lacks exonuclease activity, and
wherein the first type of nucleotide includes a detectable moiety;
detecting the first type of transiently-bound nucleotide; and
identifying the first type of transiently-bound nucleotide.

22 . The method of claim 21 , further comprising: removing the first type of nucleotide that is transiently bound to the first polymerase.

23 . The method of claim 22 , further comprising: contacting the first immobilized complex with a second type of nucleotide under suitable conditions for the first polymerase to polymerize the second type of nucleotide in a template-dependent manner.

24 . The method of claim 23 , further including polymerizing the second type of nucleotide using the first polymerase.

25 . The method of claim 23 , wherein the first type and second type of nucleotide are different types.

26 . The method of claim 23 , wherein the first type and second type of nucleotide are the same type.

27 . The method of claim 21 , wherein the detectable moiety of the first type of nucleotide includes a fluorescent label.

28 . The method of claim 21 , wherein the detectable moiety is linked to the base of the first nucleotide or linked to the terminal phosphate group of the first nucleotide.

29 . The method of claim 21 , wherein the first type of nucleotide includes an energy transfer acceptor moiety.

30 . The method of claim 21 , wherein the first polymerase includes an energy transfer donor moiety.

31 . The method of claim 21 , wherein the first nucleotide produces a FRET signal when transiently bound to the first polymerase.

32 . The method of claim 31 , wherein the FRET signal is optically detectable.

33 . The method of claim 21 , wherein the cation is present in a sufficient amount to inhibit incorporation of the first nucleotide by the first polymerase.

34 . The method of claim 21 , wherein the cation includes calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.

35 . The method of claim 21 , wherein the first polymerase comprises an RB69 polymerase, phi29 polymerase, B103 polymerase, or Klenow fragment polymerase.

36 . The method of claim 21 , wherein the first polymerase is an RB69 polymerase according to SEQ ID NO:5.

Assignees

• Life Technologies Corp

Inventors

• Vander Horn Peter
• Chen Cheng-Yao
• Luo Guobin
• Previte Michael
• Temirov Jamshid
• Nikiforov Theo
• Zhou Zhaohui
• Sun Hongye
• Wang Yufang
• Nishimura Stefanie Yukiko
• Wang Hongyi
• Peris Marian
• Rosenblum Barnett
• Phelan Michael

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6872

  involving mass spectrometry · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title