Short cycle methods for sequencing polynucleotides

• US 20050100932A1
• Filed: 05/24/2004
• Published: 05/12/2005
• Est. Priority Date: 11/12/2003
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Phillip Richard, Harris, Timothy, Lapidus, Stanley
• Granted Patent: US 7,169,560 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/113 Time

Abstract

The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.

Claims

1. A method for sequencing a nucleic acid template, the method comprising the steps of:

(a) exposing a nucleic acid template to a primer capable of hybridizing to said template and a polymerase capable of catalyzing nucleotide addition to said primer;

(b) adding a labeled nucleotide for a predetermined time, said predetermined time being coordinated with an amount of polymerization inhibition such that on average only 0, 1, or 2 labeled nucleotides are added to said primer;

(c) removing excess labeled nucleotide;

(d) neutralizing label in any incorporated nucleotide;

(e) repeating steps a, b, c, and d at least once; and

(f) determining a sequence of said template based upon the order of incorporation of said labeled nucleotides.

16. The method of claim 1 wherein said method does not utilize a blocking moiety.

17. The method of claim 1 wherein said period of time is concluded by washing said nucleotides not incorporated into said complementary strand.

18. The method of claim 1 wherein said period of time is concluded by washing said polymerization agent.

19. The method of any of claim 1 wherein said period is no more than 5 half-lives of said incorporation reactions.

20. The method of claim 1 wherein said period is no more than 4 half-lives of said incorporation reactions.

21. The method of claim 1 wherein said period is no more than 3 half-lives of said incorporation reactions.

22. The method of claim 1 wherein said period is no more than 2 half-lives of said incorporation reactions.

23. The method of claim 1 wherein said period is no more than 1 half-lives of said incorporation reactions.

24. The method of claim 1 wherein said period is no more than 0.5 half-lives of said incorporation reactions.

25. The method of claim 1 wherein said period permits less than 5% chance of incorporation of more than two of said nucleotides into said complementary strand.

26. The method of claim 1 wherein said period is no more than 1 half-life of said incorporation reactions and said wash cycles is repeated at least 40 times.

2. A method for conducting a nucleic acid sequencing reaction, the method comprising the steps of:

providing a nucleic acid template and a primer capable of hybridizing to a portion of said template, thereby to form a primed template;

exposing said primed template to a nucleotide for a period of time that is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation of said nucleotide into said primer;

detecting incorporation of said nucleotide;

neutralizing label in an incorporated nucleotide;

repeating said providing, exposing, detecting, and neutralizing steps at least once; and

determining a sequence of said template based upon the order of nucleotides incorporated into said primer.

15. The method of claim 2, further comprising

adding a first labeled nucleotide under conditions that optimize the incorporation of one of said first nucleotide per primer strand;

removing unincorporated first labeled nucleotide;

detecting any incorporated first labeled nucleotide;

neutralizing label in said first labeled nucleotide; and

adding a second labeled nucleotide under conditions that optimize the incorporation of one of said second nucleotides per primer strand.

3. A method for identifying a nucleotide incorporated into a primer in template-dependent nucleic acid sequencing, the method comprising the steps of:

conducting a plurality of base incorporation cycles, wherein each cycle comprises exposing a template nucleic acid to a labeled nucleotide that is not a chain-terminating nucleotide, wherein said labeled nucleotide is incorporated into a primer hybridized to said template if said nucleotide is capable of hybridizing to a template nucleotide immediately upstream of said primer, and wherein there is about a 99% probability that two or fewer of said nucleotides are incorporated into the same primer strand per cycle; and

identifying incorporated nucleotides.

4. A method for template-dependent nucleic acid sequencing, the method comprising the steps of:

(a) exposing a template nucleic acid to a labeled nucleotide under conditions that allow incorporation of said nucleotide into a primer attached to said template;

(b) removing unhybridized nucleotide from said template at a time after said exposing step that is sufficient for incorporation of no more than about two of said nucleotides per template;

(c) determining if a nucleotide is incorporated into said primer;

(d) identifying any incorporated nucleotide;

(e) repeating steps a, b, c, and d; and

(f) compiling a sequence of said template based upon the sequence of nucleotides incorporated into said primer.

5. A method for template-dependent nucleic acid sequencing, the method comprising the steps of:

(a) exposing a template nucleic acid to a labeled nucleotide under conditions that allow incorporation of said nucleotide into a primer attached to said template;

(b) removing unhybridized nucleotide at a time after said exposing step that is statistically insufficient for incorporation of a greater number of nucleotides than are individually optically resolvable during a predetermined detection period;

(c) detecting incorporation of individual labeled nucleotides during said detection period;

(d) neutralizing label present in incorporated nucleotides;

(e) repeating steps a, b, c, and d at least once; and

(f) compiling a sequence of said template based upon an order of incorporated nucleotides.

6. A method for nucleic acid sequencing, the method comprising the steps of:

(a) selecting a nucleic acid template to be sequenced;

(b) exposing said template to a primer that is capable of hybridizing to a portion of said template to form a primed template;

(c) selecting a desired number of nucleotides to be added to said primer;

(d) determining a reduction in the rate at which a second nucleotide is added to said primer given that a first labeled nucleotide has already been added to said primer;

(e) identifying a tolerable rate of erroneous detection of an incorporated nucleotide;

(f) exposing said primed template to a labeled nucleotide

(g) removing unincorporated labeled nucleotide at a time after said exposing step that is determined based upon said desired number, said rate reduction, and said tolerable error, such that said time is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation of said nucleotide into said primer;

(h) identifying incorporated nucleotide;

(i) neutralizing label present in said incorporated nucleotide;

(j) repeating steps f, g, and h at least once; and

(k) determining a sequence of said template based upon an order of said incorporated nucleotides.

7. A method for sequencing a template nucleic acid, the method comprising the steps of:

(a) conducting a cycle of template-dependent nucleic acid primer extension in the presence of a polymerase and a labeled nucleotide;

(b) inhibiting polymerase activity such that it is statistically unlikely that more than about 2 nucleotides are incorporated into the same primer strand in said cycle;

(c) washing unincorporated labeled nucleotide away from said template;

(d) detecting any incorporation of said labeled nucleotide;

(e) neutralizing label in any incorporated labeled nucleotide;

(f) removing said inhibition;

(g) repeating steps a, b, c, d, e, and f; and

(h) compiling a sequence of said template based upon the sequence of nucleotides incorporated into said primer.

8. A method for sequencing a target nucleic acid, the method comprising the steps of:

conducting a plurality of primer extension cycles, wherein each cycle comprises the steps of

exposing a target nucleic acid to a primer capable of hybridizing to said target thereby to form a primed target,

exposing said primed target to a labeled nucleotide in the presence of a nucleic acid polymerase,

coordinating transient inhibition of said polymerase and time of exposure to said labeled nucleotide such that it is statistically likely that at least one of said labeled nucleotide is incorporated in said primer, but statistically unlikely that more than two of said labeled nucleotide are incorporated in said primer.

9. A method for identifying a nucleotide incorporated into a primer in a template-dependent primer extension reaction, the method comprising the steps of:

exposing a template nucleic acid to a primer capable of hybridizing to said template and a polymerase capable of catalyzing template-dependent nucleotide addition to said primer;

adding a labeled nucleotide;

optically detecting whether said labeled nucleotide is incorporated into said primer, wherein said detecting occurs at a rate sufficient to detect 1, but no more than 2, incorporated nucleotides per detection cycle; and

identifying an incorporated nucleotide.

10. A method for determining the sequence of a template nucleic acid, the method comprising the steps of:

(a) exposing a nucleic acid template to a primer capable of hybridizing to a portion of said template in order to form a template/primer complex reaction mixture;

(b) adding a labeled nucleotide in the presence of a polymerase to said mixture under conditions that promote incorporation of said nucleotide into said primer if said nucleotide is complementary to a nucleotide in said template that is downstream of said primer;

(c) coordinating removal of said labeled nucleotide and inhibition of said polymerase so that no more than about 2 nucleotides are incorporated into the same primer;

(d) identifying labeled nucleotide that has been incorporated into said primer;

(e) repeating steps a, b, c, and d at least once; and

(f) determining a sequence of said template based upon the order of said nucleotides incorporated into said primer.

11. A method for identifying a nucleotide present in a template sequence, the method comprising the steps of:

exposing a template nucleic acid to a primer capable of hybridizing to a portion of said template upstream of a region of said template to be sequenced;

introducing a labeled nucleic acid and a polymerase to said template under conditions wherein said labeled nucleic acid will be incorporated in said primer if said labeled nucleic acid is capable of hybridizing with a base downstream of said primer; and

controlling the rate of said incorporation by limiting the time of exposure of said labeled nucleic acid to said template or by inhibiting said polymerase at a predefined time after exposure of said template to said labeled nucleotide;

detecting incorporation of said labeled nucleotide into said primer; and

identifying said nucleotide in said template as the complement of labeled nucleotide incorporated into said primer.

12. A method for sequencing a target nucleic acid, the method comprising the steps of:

hybridizing a nucleic acid primer comprising a donor fluorophore to a target nucleic acid at a primer binding site in said target;

exposing said hybridized primer to a first nucleotide comprising an acceptor fluorophore that, when incorporated into said primer, prevents further polymerization of said primer;

detecting the presence or absence of fluorescent emission from each of said donor and said acceptor;

identifying a nucleotide that has been incorporated into said primer via complementary base pairing with said target as the presence of fluorescent emission from said acceptor;

identifying a sequence placeholder as the absence of fluorescent emission from said donor and said acceptor; and

repeating said exposing, detecting, and each of said identifying steps, thereby to compile a sequence of said target nucleic acid based upon the sequence of said incorporated nucleotides and said placeholders.

13. A method for identifying a placeholder in a nucleic acid sequence determined by synthesis, the method comprising the steps of:

hybridizing a nucleic acid primer comprising a donor fluorophore to a target nucleic acid at a primer binding site in said target;

exposing said hybridized primer to a first nucleotide comprising an acceptor fluorophore that, when incorporated into said primer, prevents further polymerization of said primer;

determining whether there is fluorescent emission from said donor and said acceptor; and

identifying a placeholder in said nucleic acid sequence as the absence of emission in both said donor and said acceptor.

14. A method for sequencing a nucleic acid, the method comprising the steps of:

exposing a template-bound nucleic acid primer to a nucleotide comprising a label that impedes progress of polymerase in the addition of a subsequent nucleotide;

determining whether said first labeled nucleotide has been incorporated into said primer;

exposing said primer to an unlabeled first nucleotide if said first labeled nucleotide has been incorporated into said primer;

repeating said exposing and determining steps with a second nucleotide if said first nucleotide did not incorporate into said primer.