Reversible terminator molecules and methods of their use

• US 10,059,986 B2
• Filed: 04/06/2017
• Issued: 08/28/2018
• Est. Priority Date: 02/28/2013
• Status: Expired due to Fees

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Zhou, Wei, McGall, Glenn, Jain, Moti, Mei, Rui
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/481,194
• Publication Number:
  US 20170218441A1
• Time in Patent Office: 509 Days
• Field of Search: 435 61, 435 612
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C12Q 1/6869 Methods for sequencing

Abstract

The present invention relates generally to methods of sequencing of polynucleotides and compounds, compositions and kits useful for sequencing of polynucleotides. The chemical compounds include nucleotide and nucleoside analogs which possess a blocking group covalently attached to the 3′ hydroxyl of the sugar moiety. The blocking group may optionally be additionally covalently attached to a linker and/or detectable label. The nucleotide analogs may be ribonucleotide or deoxyribonucleotide analogs. Methods include incorporation of the reversible terminator molecules into growing polynucleotide strands by polymerase enzymes, such as in single base sequencing methodologies utilizing sequential reversible termination techniques.

Claims

1. A method of sequencing a polynucleotide, which comprises:

performing a polymerization reaction in a reaction system comprising a target polynucleotide to be sequenced, one or more polynucleotide primers which hybridize with the target polynucleotide to be sequenced, a catalytic amount of a polymerase enzyme and at least one reversible terminator compound selected from the group consisting of Formula;

2. The method according to claim 1, wherein Q1-Q3 are independently one or more of the following:

3. The method according to claim 1, wherein D1 and/or D2 are fluorescent detectable labels.

4. The method according to claim 1, wherein D1 and D2 are acceptor-donor FRET pair labels.

5. The method according to claim 1, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.

6. The method according to claim 1, wherein the at least one reversible terminator compound comprises a terminator compound of Formula II.

7. The method according to claim 1, wherein the at least one reversible terminator compound comprises a terminator compound of Formula III.

8. A method of sequencing a polynucleotide, which comprises:

performing a polymerization reaction in a reaction system comprising a target polynucleotide to be sequenced, one or more polynucleotide primers which hybridize with the target polynucleotide to be sequenced, a catalytic amount of a polymerase enzyme and at least one reversible terminator compound selected from the group consisting of Formula;

9. The method according to claim 8, wherein K is a halogen.

10. The method according to claim 8, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.

11. The method according to claim 8, wherein D1 and/or D2 are fluorescent detectable labels.

12. The method according to claim 8, wherein D1 and D2 are acceptor-donor FRET pair labels.

13. The method according to claim 8, wherein the at least one reversible terminator compound comprises a terminator compound of Formula II.

14. The method according to claim 8, wherein the at least one reversible terminator compound comprises a terminator compound of Formula III.

15. A method of sequencing a polynucleotide, which comprises:

performing a polymerization reaction in a reaction system comprising a target polynucleotide to be sequenced, one or more polynucleotide primers which hybridize with the target polynucleotide to be sequenced, a catalytic amount of a polymerase enzyme and at least one reversible terminator compound selected from the group consisting of Formula;

16. The method according to claim 15, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.

17. The method according to claim 15, wherein D1 and/or D2 are fluorescent detectable labels.

18. The method according to claim 15, wherein D1 and D2 are acceptor-donor FRET pair labels.

19. The method according to claim 15, wherein the at least one reversible terminator compound comprises a terminator compound of Formula I.

20. The method according to claim 15, wherein the at least one reversible terminator compound comprises a terminator compound of Formula II.