Nucleotide transient binding for sequencing methods

Patent metadata

Field	Value
Publication number	US-9255258-B2
Application number	US-201314108166-A
Country	US
Kind code	B2
Filing date	Dec 16, 2013
Priority date	Jun 5, 2009
Publication date	Feb 9, 2016
Grant date	Feb 9, 2016

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 system comprising: a polymerase which is bound with a template nucleic acid molecule that is bound to a polymerization initiation site, and a nucleotide 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, where the cation is present at a concentration that inhibits nucleotide incorporation by the polymerase, and where the polymerase is a mutant RB69 polymerase comprising the amino acid sequence of SEQ ID NO:5.

2. The system of claim 1 , 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.

3. The system of claim 1 , wherein the template nucleic acid molecule, polymerase or polymerization initiation site is linked to a surface.

4. The system of claim 1 , wherein the template nucleic acid molecule is a DNA, RNA, or DNA/RNA hybrid molecule.

5. The system of claim 1 , wherein the nucleotide includes an optically detectable label.

6. The system of claim 1 , further comprising an excitation source that emits electromagnetic radiation.

7. The system of claim 1 , wherein the polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or a reverse transcriptase.

8. The system of claim 1 , wherein the nucleotide comprises 3-10 phosphate groups.

9. The system of claim 1 , wherein the nucleotide comprises a fluorescent label linked to the base of the nucleotide or linked to the terminal phosphate group of the nucleotide.

10. The system of claim 9 , wherein the fluorescent label comprises an energy transfer acceptor reporter moiety.

11. The system of claim 1 , further comprising at least two different types of nucleotides.

12. The system of claim 11 , wherein each different type of nucleotide includes a different type of label.

13. The system of claim 1 , wherein the polymerase comprises an energy transfer donor reporter moiety.

14. The system of claim 13 , wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

15. The system of claim 1 , 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.

16. The system of claim 15 , wherein the transiently-bound labeled nucleotide produces a FRET signal.

17. The system of claim 16 , wherein the signal from the transiently-bound labeled nucleotide is optically detectable.

18. The system of claim 1 , wherein the template nucleic acid molecule includes no more than a single nucleic acid molecule.

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

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q1/6872

  involving mass spectrometry · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title