Compositions and methods for sequencing nucleic acids

• US 9,670,243 B2
• Filed: 05/27/2011
• Issued: 06/06/2017
• Est. Priority Date: 06/02/2010
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Chang, Shang-Chia, Fu, Tsu-Ju, Chiou, Chung-Fan, Pan, Chao-Chi, Fann, Jenn-Yeh
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/117,565
• Publication Number:
  US 20110300534A1
• Time in Patent Office: 2,202 Days
• Field of Search: 435 61, 435 91, 435 911, 536 41, 536 231, 536 266
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

Embodiments relate to methods of sequencing nucleic acids. Embodiments encompass the use of nucleotide analogs and a nucleic acid polymerase enzyme or enzyme complex comprising proofreading activity. The nucleotide analogs may become incorporated into a replicating strand and induce the proofreading activity of the polymerizing enzyme, thereby prolonging the duration of a signal associated with nucleotide incorporation, resulting in more observable sequencing events and increasing the accuracy of nucleic acid sequencing.

Claims

1. A method of determining the nucleotide sequence of a target nucleic acid sequence, comprising the steps of

(a) providing a reaction complex comprising a template nucleic acid comprising a target nucleic acid sequence, a primer nucleic acid comprising a. sequence which is complementary to a region of the template nucleic acid, and a polymerase enzyme or an enzyme complex, which comprises 5′

to 3′

polymerization activity and 3′

to 5′

exonuclease activity;

(b) contacting the reaction complex with a plurality of nucleotide analogs, Wherein at least one nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one non-complementary nucleotide residue and at least one label moiety comprising a photo-detectable label that is indicative of the identity of the base-pairing moiety, further wherein the at least one nucleotide analog of said plurality is chosen from compounds having formula I;

2. The method of claim 1, wherein the photo-detectable label is a fluorophore.

3. The method of claim 2, wherein at least one of said plurality of nucleotide analogs comprises at least one fluorescence quenching moiety, and wherein the fluorescence quenching moiety is removed from the nucleotide analog upon incorporation of the nucleotide analog via the enzyme'"'"'s or enzyme complex'"'"'s 5′

to 3′

polymerization activity into the nascent strand.

4. The method of claim 1, wherein the at least one base-pairing moiety, having a 5′

end and a 3′

end, comprises one or more nucleotide residues that each comprises a base that is able to base pair with a corresponding base of the target nucleic acid sequence in an active site of the reaction complex.

5. The method of claim 4, wherein the 3′

end of the base-pairing moiety is connected to the label moiety via a phosphate linkage.

6. The method of claim 3, wherein the fluorescence quenching moiety is attached to the 5′

end of a nucleotide analog.

7. The method of claim 6, wherein the fluorescence quenching moiety is attached to the 5′

end of the nucleotide analog via a linker.

8. The method of claim 6, wherein the nucleotide analog is a nucleotide triphosphate analog having an alpha phosphate, a beta phosphate and a gamma phosphate, and wherein the fluorescence quenching moiety is attached to the beta or gamma phosphate of the triphosphate group at the 5′

end of the nucleotide analog.

9. The method of claim 4, wherein the at least one base-pairing moiety comprises at least three phosphate groups at its 5′

end, wherein the phosphate group most proximal to the base-pairing moiety (the alpha phosphate) is a phosphorothioate.

10. The method of claim 4, wherein the at least one base-pairing moiety comprises at least three phosphate groups at its 5′

end, wherein the phosphate group most proximal to the base-pairing moiety (the alpha phosphate) is a methylphosphonate.

11. The method of claim 4, Wherein. the at least one base-pairing moiety comprises at least three phosphate groups at its 5′

end, wherein the phosphate group most proximal to the base-pairing moiety (the alpha phosphate) is a boranophosphate.

12. The method of claim 1, wherein the length of time between binding of the nucleotide analog at the active site of the reaction complex and removal of the label moiety from the nascent strand is from about 50 to 250 milliseconds.

13. The method of claim 1, wherein the method is performed under conditions whereby the length of time between two successive detecting steps (d) is from about 0.2 seconds to 1 second.

14. The method of claim 1, wherein the method is performed under conditions whereby the length of time between two successive detecting steps (d) is from about 0.2 to 0.6 seconds.

15. The method of claim 1, wherein the method is performed under conditions whereby the length of time between two successive detecting steps (d) is from about 0.3 to 0.5 seconds.

16. The method of claim 1, wherein repeating steps (c)-(e) to determine the sequence of the target nucleic acid in step (f) comprises continuously monitoring and recording the sequential incorporation of each photo-detectable label in the label moiety of nucleotide analogs being sequentially incorporated into the primer strand.

17. The method of claim 1, wherein n is 1, R₁ is

18. The method of of claim 1, wherein Y₁ is S⁻

.

19. The method of claim 1, wherein R₃ is

20. The method of claim 1, wherein Y₃ is O⁻

.

21. The method of claim 1, wherein the plurality of nucleotide analogs comprises at least four types of nucleotide analogs, each type comprising a unique base-pairing moiety and a unique fluorophore that is distinguishable from the fluorophores of the other types of nucleotide analogs of said plurality.

22. The method of claim 21, wherein the plurality of nucleotide analogs further comprises at least one nucleotide analog having a fluorescence quenching moiety that is removed from the nucleotide analog upon incorporation of the nucleotide analog into the nascent strand.

25. The method of claim 1 or 23, wherein the target nucleic acid sequence comprises sequence repeats.

26. The method of claim 1 or 23, wherein the target nucleic acid sequence comprises one or more sequence repeats selected from (A)n, (T)n, (C)n, or (G)n, wherein n is an integer of 3 or greater.

27. The method of claim 1, wherein R₅ is H.

23. A method of determining a nucleotide base incorporated by a polymerase enzyme or enzyme complex in a nucleic acid polymerization reaction, the method comprising the steps of:

(a) conducting a nucleic acid polymerization reaction that utilizes both 5′

to 3′

polymerization activity and 3 to 5′

exonuclease activity of a polymerase enzyme or enzyme complex, and that results in production of a nascent strand in a template-dependent manner, Wherein said reaction is conducted in the presence of

(i) a template nucleic acid comprising a target nucleic acid sequence,

(ii) a primer nucleic acid comprising a sequence which is complementary to a region of the template nucleic acid,

(iii) a polymerase enzyme or an enzyme complex comprising 5′

to 3′

polymerization activity and 3′

to 5′

exonuclease activity,

(iv) a plurality of nucleotide analogs, wherein at least one nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one non complementary nucleotide residue, and at least one label moiety, said label moiety comprising a photo-detectable label, wherein the at least one nucleotide analog of said plurality is chosen from compounds having formula I;

24. The method of claim 23, wherein said detecting step is performed during each successive nucleotide incorporation event after the photo-detectable label of the nucleotide analog incorporated in the previous incorporation event is removed via the 3′

to 5′

exonuclease activity of the enzyme or enzyme complex.

28. A method of determining the nucleotide sequence of a target nucleic, acid sequence, comprising the steps of:

(a) providing a reaction complex comprising a template nucleic acid comprising a target nucleic acid sequence, a primer nucleic acid comprising a sequence which is complementary to a. region of the template nucleic acid, and a polymerase enzyme or an enzyme complex, which comprises 5′

to 3′

polymerization activity and 3′

to 5′

exonuclease activity;

(b) contacting the reaction complex with a plurality of nucleotide analogs;

(c) allowing the enzyme or enzyme complex to incorporate a nucleotide analog in a template-dependent manner into a nascent strand via 5′

to 3′

polymerization activity of the enzyme or enzyme complex;

(d) detecting the photo-detectable label moiety of the incorporated nucleotide analog;

(e) allowing the enzyme or enzyme complex to remove the label moiety of the incorporated nucleotide analog from the nascent strand via 3′

to 5′

exonuclease activity of the enzyme or enzyme complex; and

(f) repeating steps (c)-(e) to determine the sequence of the target nucleic acid sequence,

wherein at least one of the nucleotide analogs is chosen from compounds having formula I;