Methods and apparatus that increase sequencing-by-binding efficiency
A method of nucleic acid detection including (a) contacting a primed template with a polymerase, nucleotide cognate of a first base type and nucleotide cognate of a second base type under ternary complex stabilizing conditions; (b) contacting the primed template nucleic acid with a polymerase, nucleotide cognate of the first base type and nucleotide cognate of a third base type under ternary complex stabilizing conditions; (c) examining products of (a) and (b) for signals produced by a ternary complex that includes the primed template nucleic acid, polymerase and next correct nucleotide, wherein signals for the product of (a) are ambiguous for the first and second base type, and signals for the product of (b) are ambiguous for the first and third base type; (d) disambiguating signals acquired in (c) to identify the next correct nucleotide.
1. A method of nucleic acid detection, comprising steps of:
(a) contacting a primed template nucleic acid with a polymerase and a first mixture of nucleotides under conditions for forming a ternary complex at a nucleotide position in the template, wherein the first mixture comprises
a nucleotide cognate of a first base type comprising a label that produces a first signal;
a nucleotide cognate of a second base type comprising a label that produces the first signal;
a nucleotide cognate of a third base type comprising a label that produces a second signal; and
a nucleotide cognate of a fourth base type comprising a label that produces the second signal;
(b) contacting the primed template nucleic acid with a polymerase and a second mixture of nucleotides under conditions for forming a ternary complex at the nucleotide position in the template, wherein the second mixture comprises
a nucleotide cognate of the first base type comprising a label that produces the first signal;
a nucleotide cognate of a second base type comprising a label that produces the second signal;
a nucleotide cognate of a third base type comprising a label that produces the second signal; and
a nucleotide cognate of the fourth base type comprising a label that produces the first signal; and
(c) examining products of steps (a) and (b) for signals produced by a ternary complex that comprises the primed template nucleic acid, a polymerase and a next correct nucleotide.
2. The method of claim 1 , wherein the signals for the products of step (a) are acquired by a detector that is also used to detect the signals for the products of step (b).
3. The method of claim 1 , further comprising (d) adding a reversibly terminated, next correct nucleotide to the primer of the primed template nucleic acid after step (c), thereby producing an extended, reversibly terminated primer.
4. The method of claim 3 , further comprising repeating steps (a) through (c) for the primed template nucleic acid that comprises the extended, reversibly terminated primer.
5. The method of claim 4 , further comprising (e) removing the reversible terminator moiety from the extended, reversibly terminated primer after steps (a) through (c) are repeated.
6. The method of claim 1 , wherein the steps are carried out for a plurality of primed template nucleic acids having different sequences.
7. The method of claim 6 , wherein the plurality of primed template nucleic acids is attached to an array.
8. The method of claim 1 , wherein the examining of the products of step (a) is carried out prior to step (b).
9. The method of claim 1 , further comprising
(i) contacting the primed template nucleic acid with a polymerase and a third mixture of nucleotides under conditions for forming a ternary complex at the nucleotide position in the template, wherein the third mixture comprises a nucleotide cognate of the second base type and a nucleotide cognate of a fourth base type;
(ii) contacting the primed template nucleic acid with a polymerase and a fourth mixture of nucleotides under conditions for stabilizing a ternary complex at the nucleotide position in the template, wherein the fourth mixture comprises a nucleotide cognate of the third base type and a nucleotide cognate of the fourth base type; and
(iii) examining products of steps (i) and (ii) for signals produced by a ternary complex that comprises the primed template nucleic acid, a polymerase and a next correct nucleotide.
10. A method of nucleic acid detection, comprising:
(a) sequentially contacting a primed template nucleic acid with first and second mixtures under conditions for forming a ternary complex, wherein each of the mixtures comprises a polymerase and nucleotide cognates for at least two of four different base types in the primed template nucleic acid, wherein the first mixture comprises
a nucleotide cognate of a first base type comprising a label that produces a first signal;
a nucleotide cognate of a second base type comprising a label that produces the first signal;
a nucleotide cognate of a third base type comprising a label that produces a second signal; and
a nucleotide cognate of a fourth base type comprising a label that produces the second signal; and the second mixture comprises
a nucleotide cognate of the first base type comprising a label that produces the first signal;
a nucleotide cognate of a second base type comprising a label that produces the second signal;
a nucleotide cognate of a third base type comprising a label that produces the second signal; and
a nucleotide cognate of the fourth base type comprising a label that produces the first signal;
(b) examining the first and second mixtures, or products thereof, separately to detect ternary complexes; and
(c) identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein the next correct nucleotide is identified as a cognate of one of the four different base types if ternary complex is detected in the two mixtures.
11. The method of claim 10 , wherein step (a) comprises sequentially contacting the primed template nucleic acid with at least three mixtures under conditions for forming a ternary complex, wherein each of the mixtures comprises a polymerase and nucleotide cognates for four different base types in the primed template nucleic acid, wherein the mixtures differ by at least one type of nucleotide cognate.
12. The method of claim 11 , wherein the next correct nucleotide is identified as a cognate of one of the four different base types if ternary complex is detected in at least two of the mixtures.
13. The method of claim 1 , wherein the first and second signals comprise optical signals.
14. The method of claim 13 , wherein the first and second signals examined in step (c) are distinguished by different luminescence emission wavelengths.
15. The method of claim 13 , wherein the first and second signals examined in step (c) are distinguished by different luminescence excitation wavelengths.
16. The method of claim 13 , wherein the first and second signals examined in step (c) are distinguished by different luminescent intensities at a wavelength.
17. The method of claim 5 , further comprising repeating steps (a) through (e) to determine the nucleotide sequence for the template nucleic acid.
18. The method of claim 10 , further comprising (d) adding a reversibly terminated, next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby producing an extended, reversibly terminated primer.
19. The method of claim 18 , further comprising repeating steps (a) and (b) for the primed template nucleic acid that comprises the extended, reversibly terminated primer.
20. The method of claim 19 , further comprising (e) removing the reversible terminator moiety from the extended, reversibly terminated primer after steps (a) and (b) are repeated.
21. The method of claim 18 , wherein step (d) is carried out prior to step (c).
22. The method of claim 10 , wherein the steps are carried out for a plurality of primed template nucleic acids having different sequences.
23. The method of claim 22 , wherein the plurality of primed template nucleic acids is attached to an array.