NUCLEOTIDE TRANSIENT BINDING FOR SEQUENCING METHODS

• US 20170369857A1
• Filed: 08/30/2017
• Published: 12/28/2017
• 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
• Granted Patent: US 10,597,642 B2
• Time in Patent Office: Days
• 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 method for sequencing a nucleic acid molecule, comprising:

a) transiently binding, in a template-dependent manner and without polymerizing, a first type of nucleotide to a first polymerase in the presence of a metal cation, wherein the metal cation inhibits nucleotide incorporation by the polymerase, wherein the first polymerase is bound to a template nucleic acid molecule that is bound to a polymerization initiation site, wherein the first polymerase lacks exonuclease activity, and wherein the first type of nucleotide includes a detectable moiety;

b) detecting the first type of transiently-bound nucleotide;

c) identifying the first type of transiently-bound nucleotide;

d) removing the transiently-bound first type of nucleotide from the first polymerase;

e) contacting the first polymerase with a second type of nucleotide under suitable conditions for the first polymerase to polymerize the second type of nucleotide at the polymerization initiation site in a template-dependent manner; and

f) repeating steps (a)-(e) at least once.

2. The method of claim 1, wherein the suitable conditions in step (e) further comprise any one or more of the following:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a metal cation that inhibits nucleotide incorporation;

(ii) the first polymerase selectively binds the second type of nucleotide in a template-dependent manner and polymerizes the bound second type of nucleotide;

(iii) the second type of nucleotide is an incorporatable nucleotide; and

(iv) the polymerization initiation is an extendible polymerization initiation site.

3. The method of claim 1, wherein the metal cation that inhibits nucleotide incorporation is calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.

4. The method of claim 1, wherein the first type of nucleotide in step (a) includes an adenosine, guanosine, cytidine, thymidine, or uridine moiety.

5. The method of claim 1, wherein the detectable moiety includes an optically detectable label, and wherein the method further includes exciting the optically detectable label with electromagnetic radiation.

6. The method of claim 1, wherein the first type of nucleotide in step (a) comprises 3-10 phosphate groups.

7. The method of claim 1, wherein the first type of nucleotide comprises a fluorescent dye linked to the base of the nucleotide or a fluorescent dye linked to the terminal phosphate group of the nucleotide.

8. The method of claim 1, wherein the detectable moiety comprises an energy transfer acceptor reporter moiety.

9. The method of claim 1, wherein the first polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or a reverse transcriptase.

10. The method of claim 1, wherein the first polymerase is an RB69 polymerase, phi29 polymerase, B103 polymerase, or Klenow fragment polymerase.

11. The method of claim 1, wherein the first polymerase is an RB69 polymerase according to SEQ ID NO:

5.

12. The method of claim 1, wherein the first polymerase comprises an energy transfer donor reporter moiety.

13. The method of claim 12, wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

14. The method of claim 1, wherein the first polymerase comprises an energy transfer donor reporter moiety and the first type of nucleotide comprises an energy transfer acceptor reporter moiety, and wherein the first nucleotide transiently-bound to the first polymerase produces a FRET signal.

15. The method of claim 14, wherein the signal from the transiently-bound labeled nucleotide is optically detectable.

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

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