REVERSIBLE DI-NUCLEOTIDE TERMINATOR SEQUENCING

• US 20090263797A1
• Filed: 11/20/2008
• Published: 10/22/2009
• Est. Priority Date: 11/20/2007
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: ZON, Gerald
• Granted Patent: US 8,017,338 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides

Abstract

The present teachings provide methods, compositions, and kits for synthesizing and sequencing nucleic acids. In some embodiments, reversible di-nucleotide compounds are employed along with cleaving reactions that remove a label and a blocking moiety. Improved sequencing efficiency is achieved by the rapid polymerase-mediated incorporation of reversible di-nucleotide compounds. In some embodiments, the di-nucleotides do not contain conventional nucleotide triphosphates, but rather employ amino acid phosphoramidate nucleotides (AAPNs).

Claims

1. A method for determining a sequence of nucleotides in a template, the method comprising:

(a) hybridizing a primer to a template;

(b) extending the primer in the presence of a reversible di-nucleotide terminator, wherein the reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide;

(c) identifying a family of the reversible di-nucleotide terminator;

(d) generating an extendable terminus on the incorporated reversible di-nucleotide terminator;

(e) repeating steps (b), (c), and (d) a suitable number of times to form an extension product hybridized to the template;

(f) removing the extension product;

(g) hybridizing a shifted-primer to the template;

(h) extending the primer in the presence of a reversible di-nucleotide terminator, wherein the reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide;

(i) identifying a family of the reversible di-nucleotide terminator;

j) generating an extendable terminus on the incorporated reversible di-nucleotide terminator;

(k) repeating steps (h), (i), and (j) a suitable number of times; and

,

(l) sequencing the template.

2. The method according to claim 1 wherein the reversible di-nucleotide terminator has a blocking moiety at a 3′

position on the second nucleotide.

3. The method according to claim 1 wherein the identifying includes detecting a label attached to the most recently extended reversible di-nucleotide terminator.

4. The method according to claim 1 further including a capping of an extension product whenever no reversible di-nucleotide terminator has been extended during the extending.

5. The method according to claim 1 wherein the template polynucleotide is prepared by;

(a) amplifying the template in a compartment of an emulsion in the presence of microparticle so that a microparticle having a clonal population of template attached thereto is produced;

(b) recovering the microparticle from the emulsion; and

,

(c) embedding the microparticle in or on a semi-solid support.

12. The method according to claim 1 or claim 6 in which the reversible di-nucleotide terminator comprises an AAPN.

6. A method for determining information about a sequence of nucleotides in a template using a first collection of at least 2 distinguishably labeled reversible di-nucleotide terminators, the method comprising;

(a) extending a primer along the template by incorporating a reversible di-nucleotide terminator to form an extension product, wherein the reversible di-nucleotide terminator is a member of the collection of distinguishably labeled reversible di-nucleotide terminator;

(b) detecting a label associated with the reversible di-nucleotide terminator;

(c) repeating steps (a) and (b) until an ordered list of reversible di-nucleotide terminator family names is obtained; and

,

(d) using the ordered list of reversible di-nucleotide terminator family names to eliminate one or more possibilities for the sequence of nucleotides of the template.

7. The method according to claim 6 wherein (d) comprises decoding the ordered list of reversible di-nucleotide terminator family names to determine the sequence.

8. The method according to claim 7 wherein the decoding comprises;

generating at least one candidate sequence from the ordered list of reversible di-nucleotide terminator family names; and

,

selecting a candidate sequence as the sequence of nucleotides in the template.

9. A collection of distinguishably labeled reversible di-nucleotide terminator families, wherein each family comprises a collection of di-nucleotides, and wherein the collection of di-nucleotides in a probe family comprise the same label.

10. The collection of distinguishably labeled reversible di-nucleotide terminator families of claim 9, wherein each reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide, wherein the second nucleotide comprises a label on the base, and wherein the second nucleotide comprises a blocking moiety at the 3′

position of the second nucleotide.

11. The collection of distinguishably labeled reversible di-nucleotide terminator families of claim 9, wherein each di-nucleotide contains an internucleotide moiety that is a phosphotriester moiety.

13. A method for determining a sequence of nucleotides in a template, the method comprising:

(a) hybridizing a primer to a template;

(b) extending the primer in the presence of a reversible di-nucleotide terminator, wherein the reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide, to incorporated the reversible di-nucleotide terminator;

(c) identifying two bases in the template based on a dye associated with the incorporated di-nucleotide terminator;

(d) generating an extendable terminus on the incorporated reversible di-nucleotide terminator,

(e) repeating steps (b), (c), and (d) a suitable number of times to determine the sequence of nucleotides in the template.

14. The method according to claim 13 wherein the reversible di-nucleotide terminator comprises a blocking moiety at the 3′

position of the 3′

nucleotide.

15. The method according to claim 13 wherein the reversible di-nucleotide terminator comprises a dye attached to a base of a 3′

nucleotide.

16. The method according to claim 13 wherein the template is bead-immobilized.

17. The method according to claim 13 wherein at least one of the nucleotides in the reversible di-nucleotide terminator comprises an amino acid phosphoramidate nucleotide.

18. A kit for determining information about a sequence of nucleotides in a template polynucleotide comprising;

a collection of distinguishably labeled reversible di-nucleotide terminator families, wherein each family comprises a collection of reversible di-nucleotide terminators, and wherein the collection of di-nucleotides in a family comprise the same label; and

,

a suitable polymerase.

19. The kit according to claim 18 further comprising microparticles.

20. The kit according to claim 18 further comprising a primer and a shifted primer.

21. The kit according to claim 18 wherein at least one of the nucleotides in the reversible di-nucleotide terminator comprises an amino acid phosphoramidate nucleotide.