Methods of producing and sequencing modified polynucleotides

• US 8,058,030 B2
• Filed: 12/07/2009
• Issued: 11/15/2011
• Est. Priority Date: 06/28/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Smith, Douglas R., McKernan, Kevin
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/632,713
• Publication Number:
  US 20100285461A1
• Time in Patent Office: 708 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone

Abstract

The present invention encompasses methods for producing a modified polynucleotide sequence that comprises a (e.g., one or more) phosphorothiolate linkage, methods for determining a polynucleotide sequence comprising a (e.g., one or more) phosphorothiolate linkage, and methods for separating forward and reverse extension products that comprise a (e.g., one or more) phosphorothiolate linkage. The invention also encompasses kits for producing and/or determining the sequence of a modified polynucleotide that comprises a (e.g., one or more) phosphorothiolate linkage.

Claims

1. A method for determining a polynucleotide sequence comprising:

a) annealing a plurality of primers to a plurality of template polynucleotide sequences;

b) extending the plurality of primers in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing a plurality of extension products that comprise a modified nucleotide sequence having one or more phosphorothiolate linkages;

c) cleaving the phosphorothiolate linkages in the extension products, under conditions in which a plurality of fragments are produced;

d) identifying from among the fragments produced in c), the fragments which comprise the primer; and

e) identifying the nucleotide at the 3′

end of the fragments identified in d), thereby determining a polynucleotide sequence.

2. The method of claim 1, wherein each primer in the plurality of primers comprises a first tag.

3. The method of claim 1, wherein the one or more nucleoside triphosphates comprises a tag.

4. The method of claim 1, wherein the phosphorothiolate linkage is cleaved by Ag⁺, Hg²⁺ or Cu²⁺.

5. The method of claim 4, wherein the phosphorothiolate linkage is cleaved at a pH of about 7 and at a temperature of about 22°

C. to about 37°

C.

6. A method for determining a polynucleotide sequence comprising:

a) annealing a plurality of primers to a plurality of template polynucleotide sequences;

b) extending the plurality of primers in the presence of one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [I];

7. The method of claim 6, wherein R₁ is hydrogen (—

H) or —

SCH₃.

8. The method of claim 6, wherein each template polynucleotide sequence in the plurality of template polynucleotide sequences comprises a sense strand and an antisense strand and wherein at least one primer is annealed to each strand prior to step (b).

9. The method of claim 6, wherein each primer in the plurality of primers comprises a tag.

10. A method for determining a polynucleotide sequence comprising:

a) annealing a plurality of primers to a plurality of template polynucleotide sequences, wherein each primer in the plurality of primers comprises a first tag;

b) extending the plurality of primers in the presence of one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [III];

11. The method of claim 10, wherein each template polynucleotide sequence in the plurality of template polynucleotide sequences comprises a sense strand and an antisense strand and wherein at least one primer is annealed to each strand prior to step (b).