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 system comprising: a polymerase which is bound with a template nucleic acid molecule that is bound to a polymerization initiation site, and a nucleotide which is transiently bound to the active site of the polymerase in a template-dependent manner, and a cation that inhibits nucleotide incorporation by the polymerase, where the cation is present at a concentration that inhibits nucleotide incorporation by the polymerase, and where the polymerase is a mutant RB69 polymerase comprising the amino acid sequence of SEQ ID NO:5.
2. The system of claim 1 , wherein the cation that inhibits nucleotide incorporation is calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.
3. The system of claim 1 , wherein the template nucleic acid molecule, polymerase or polymerization initiation site is linked to a surface.
4. The system of claim 1 , wherein the template nucleic acid molecule is a DNA, RNA, or DNA/RNA hybrid molecule.
5. The system of claim 1 , wherein the nucleotide includes an optically detectable label.
6. The system of claim 1 , further comprising an excitation source that emits electromagnetic radiation.
7. The system of claim 1 , wherein the polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or a reverse transcriptase.
8. The system of claim 1 , wherein the nucleotide comprises 3-10 phosphate groups.
9. The system of claim 1 , wherein the nucleotide comprises a fluorescent label linked to the base of the nucleotide or linked to the terminal phosphate group of the nucleotide.
10. The system of claim 9 , wherein the fluorescent label comprises an energy transfer acceptor reporter moiety.
11. The system of claim 1 , further comprising at least two different types of nucleotides.
12. The system of claim 11 , wherein each different type of nucleotide includes a different type of label.
13. The system of claim 1 , wherein the polymerase comprises an energy transfer donor reporter moiety.
14. The system of claim 13 , wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.
15. The system of claim 1 , wherein the polymerase comprises a label including an energy transfer donor reporter moiety and the transiently-bound nucleotide comprises an energy transfer acceptor reporter moiety.
16. The system of claim 15 , wherein the transiently-bound labeled nucleotide produces a FRET signal.
17. The system of claim 16 , wherein the signal from the transiently-bound labeled nucleotide is optically detectable.
18. The system of claim 1 , wherein the template nucleic acid molecule includes no more than a single nucleic acid molecule.