Nucleotide transient binding for sequencing methods

• US 9,255,258 B2
• Filed: 12/16/2013
• Issued: 02/09/2016
• Est. Priority Date: 06/05/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• 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
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US14/108,166
• Publication Number:
  US 20140234853A1
• Time in Patent Office: 785 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value

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.

Claims

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.