Nucleotide transient binding for sequencing methods

Patent metadata

Field	Value
Publication number	US-12152256-B2
Application number	US-202217932844-A
Country	US
Kind code	B2
Filing date	Sep 16, 2022
Priority date	Jun 5, 2009
Publication date	Nov 26, 2024
Grant date	Nov 26, 2024

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

What is claimed:

1. A method for identifying a nucleotide bound to a polymerase, comprising:
a) contacting at least one type of a labeled nucleotide to an immobilized complex having a first polymerase bound to a template nucleic acid molecule that is bound to a polymerization initiation site, under suitable conditions to transiently-bind the at least one type of labeled nucleotide to the polymerase in a nucleic acid template-dependent manner and to inhibit nucleotide polymerization by the polymerase;
b) detecting the transiently-bound labeled nucleotide; and
c) identifying the labeled nucleotide transiently-bound to the polymerase, wherein the polymerase is a modified phi29 polymerase that includes lysine at position 379 and/or 383 substituted with leucine or arginine.

2. A system comprising:
a) a polymerase which lacks exonuclease activity complexed with a template nucleic acid molecule which is bound to a polymerization initiation site; a nucleotide including a detectable moiety which is transiently bound to the active site of the polymerase in a template-dependent manner; and a cation that inhibits nucleotide incorporation by the polymerase, present at a concentration that inhibits nucleotide incorporation by the polymerase
b) a detection system comprising excitation illumination elements, optical transmission elements and detectors,
wherein the polymerase is a modified phi29 polymerase that includes lysine at position 379 and/or 383 substituted with leucine or arginine.

3. The system of claim 2 , wherein the transiently-bound nucleotide is inhibited from incorporation onto the polymerization initiation site by suitable conditions which include: (i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation; (ii) using a polymerase which selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity; (iii) using at least one type of labeled nucleotide which is a labeled non-incorporatable nucleotide; and/or (iv) using a polymerization initiation site which is a non-extendible polymerization initiation site.

4. The system of claim 2 , wherein the cation that inhibits nucleotide incorporation is calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.

5. The system of claim 2 , which lacks a catalytic metal ion, including magnesium, manganese, cobalt, strontium, or barium.

6. The system of claim 2 , wherein the template nucleic acid molecule, polymerase or polymerization initiation site is linked to a surface.

7. The system of claim 2 , wherein the template nucleic acid molecule is a DNA, RNA, or DNA/RNA hybrid molecule.

8. The system of claim 2 , wherein the nucleotide includes an optically detectable label.

9. The system of claim 2 , further comprising an excitation source that emits electromagnetic radiation.

10. The system of claim 2 , wherein the nucleotide comprises 3-10 phosphate groups.

11. The system of claim 2 , wherein the nucleotide comprises a fluorescent label linked to the base of the nucleotide or linked to the terminal phosphate group of the nucleotide.

12. The system of claim 11 , wherein the fluorescent label comprises an energy transfer acceptor reporter moiety.

13. The system of claim 2 , further comprising at least two different types of nucleotides.

14. The system of claim 13 , wherein each different type of nucleotide includes a different type of label.

15. The system of claim 2 , wherein the polymerase comprises an energy transfer donor reporter moiety.

16. The system of claim 15 , wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

17. The system of claim 2 , wherein the polymerase comprises a label including an energy transfer donor reporter moiety and the transiently-bound nucleotide comprises an energy transfer acceptor reporter moiety.

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

• C12Y207/07007

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

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q1/6818

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

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title