Multivalent binding composition for nucleic acid analysis
Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.
1. A system comprising:
(a) a flow cell with an interior surface comprising a plurality of primed target nucleic acid sequences coupled thereto, wherein a primed target nucleic acid sequence of said plurality of primed target nucleic acid sequences has a polymerase bound thereto:
(b) a fluid flow controller configured to control sequential and iterative delivery of a reagent to said interior surface of said flow cell;
(c) an imaging module configured to image said interior surface of said flow cell; and
(d) a processor, wherein said processor is programed to instruct said system to perform an iterative method comprising:
(i) contacting said plurality of primed target nucleic acid sequences coupled to said interior surface of said flow cell with a conjugated nucleotide composition to form a binding complex between said plurality of primed target nucleic acid sequences and a plurality of nucleotide moieties of said conjugated nucleotide composition, wherein said binding complex has a persistence time of at least 2 seconds when a nucleotide moiety of said plurality of nucleotide moieties is complementary to a nucleotide of said primed target nucleic acid sequence; and
(ii) imaging said interior surface of said flow cell to detect said binding complex and thereby determine an identity of said nucleotide of said primed target nucleic acid sequence.
2. The system of claim 1 , wherein said flow cell comprises a capillary.
3. The system of claim 1 , wherein said flow cell comprises a capillary etched into a planar chip.
4. The system of claim 1 , wherein said flow cell comprises a removable or disposable component of said system.
5. The system of claim 1 , wherein said interior surface of said flow cell comprises one or more hydrophilic polymer layers.
6. The system of claim 5 , wherein at least one of said one or more hydrophilic polymer layers comprises a branched hydrophilic polymer molecule.
7. The system of claim 6 , wherein said branched hydrophilic polymer molecule comprises two or more nucleotide moieties.
8. The system of claim 1 , wherein said persistence time is at least 10 seconds.
9. The system of claim 1 , wherein said persistence time is at least 20 seconds.
10. The system of claim 1 , wherein said processor of (d) is programmed to perform a sequencing reaction cycle comprising steps (i)-(ii) in less than 60 minutes.
11. The system of claim 1 , wherein said processor of (d) is programmed to perform steps (i) to (ii) interactively to determine said target nucleic acid sequence, and wherein an average Q-score for base-calling accuracy while determining said target nucleic acid sequence is at least 30.
12. The system of claim 1 , wherein said system provides a Q-score of greater than 30 for at least 80% of a plurality of nucleotides of the plurality of primed target nucleic acid sequences identified by said system.
13. The system of claim 1 , wherein said imaging module comprises one or more light sources.
14. The system of claim 1 , wherein said conjugated nucleotide composition comprises one or more detectable labels.
15. The system of claim 14 , wherein said one or more detectable labels comprises a plurality of detectable labels.
16. The system of claim 14 , wherein said one or more detectable labels are fluorescent labels.
17. The system of claim 1 , wherein said binding complex further comprises a polymerase.
18. The system of claim 17 , wherein said polymerase is catalytically inactive.
19. The system of claim 1 , wherein said polymerase has been rendered catalytically inactive by mutation or chemical modification.
20. The system of claim 1 , wherein a primed target nucleic acid sequence of said plurality of primed target nucleic acid sequences comprises a blocked nucleotide at 3′ end thereof, wherein said blocked nucleotide comprises a blocking group.