Nucleotide transient binding for sequencing methods
Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation.
1. A method for identifying a nucleotide bound to a polymerase, comprising:
a) transiently binding in a template-dependent manner, and without polymerizing, a first type of nucleotide to a first polymerase in the presence of a divalent cation that inhibits nucleotide incorporation by the first polymerase,
wherein the first polymerase is included in a first immobilized complex comprising (i) the first polymerase, (ii) a first template nucleic acid molecule, and (iii) a first polymerization initiation site,
wherein the first immobilized complex is linked to a first site on a surface and the surface includes a plurality of immobilized complexes linked to other sites on the same surface wherein the first polymerase comprises the amino acid sequence of SEQ ID NO:5 wherein the first polymerase lacks exonuclease activity, and
wherein the first type of nucleotide includes a detectable moiety;
b) detecting the first type of transiently-bound nucleotide; and
c) identifying the first type of transiently-bound nucleotide.
2. The method of claim 1 , further comprising: removing the first type of nucleotide that is transiently bound to the first polymerase.
3. The method of claim 2 , further comprising: contacting the first immobilized complex with a second type of nucleotide under suitable conditions for the first polymerase to polymerize the second type of nucleotide in a template-dependent manner.
4. The method of claim 3 , further including polymerizing the second type of nucleotide using the first polymerase.
5. The method of claim 3 , wherein the first type and second type of nucleotide are different types.
6. The method of claim 3 , wherein the first type and second type of nucleotide are the same type.
7. The method of claim 1 , wherein the detectable moiety of the first type of nucleotide includes a fluorescent label.
8. The method of claim 1 , wherein the detectable moiety is linked to the base of the first nucleotide or linked to the terminal phosphate group of the first nucleotide.
9. The method of claim 1 , wherein the first type of nucleotide includes an energy transfer acceptor moiety.
10. The method of claim 1 , wherein the first polymerase includes an energy transfer donor moiety.
11. The method of claim 1 , wherein the first nucleotide produces a FRET signal when transiently bound to the first polymerase.
12. The method of claim 1 , wherein the divalent cation that inhibits nucleotide incorporation by the first polymerase includes calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.
13. The method of claim 3 , wherein the second detectable moiety of the second type of nucleotide includes a fluorescent label.
14. The method of claim 3 , wherein the second detectable moiety is linked to the base of the second nucleotide or linked to the terminal phosphate group of the second nucleotide.
15. The method of claim 3 , wherein the second type of nucleotide includes an energy transfer acceptor moiety.
16. The method of claim 15 , wherein the second nucleotide produces a FRET signal during the template-dependent polymerizing.