Methods and apparatus for single molecule sequencing using energy transfer detection
Provided herein are systems and methods for nucleotide incorporation reactions. The systems comprise polymerases having altered nucleotide incorporation kinetics and are linked to an energy transfer donor moiety, and nucleotide molecules linked with at least one energy transfer acceptor moiety. The donor and acceptor moieties undergo energy transfer when the polymerase and nucleotide are proximal to each other during nucleotide binding and/or nucleotide incorporation. As the donor and acceptor moieties undergo energy transfer, they generate an energy transfer signal which can be associated with nucleotide binding or incorporation. Detecting a time sequence of the generated signals, or the change in the signals, can be used to determine the order of the incorporated nucleotides, and can therefore be used to deduce the sequence of the target molecule.
1. A method for generating an energy transfer signal comprising the steps of:
contacting (i) a polymerase having altered nucleotide incorporation kinetics and linked to an energy transfer donor moiety with (ii) a nucleic acid molecule and with (iii) at least one type of a nucleotide having an energy transfer acceptor moiety, so as to incorporate the nucleotide into the nucleic acid molecule thereby locating the polymerase and nucleotide in close proximity with each other to generate the energy transfer signal, wherein the polymerase comprises the amino acid sequence of SEQ ID NO:2.
2. The method of claim 1 , further comprising the steps of:
a) exciting the energy transfer donor moiety with an excitation source; and
b) detecting the energy transfer signal from the energy transfer donor moiety and the energy transfer acceptor moiety that are in close proximity to each other.
3. The method of claim 2 , further comprising the steps of:
a) identifying the energy transfer signal from the energy transfer acceptor moiety.
4. The method of claim 1 , wherein the polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or reverse transcriptase.
5. The method of claim 1 , wherein the altered nucleotide incorporation kinetics includes altered polymerase binding to the target molecule, altered polymerase binding to the nucleotide, altered polymerase catalyzing nucleotide incorporation, altered the polymerase cleaving the phosphate group or substituted phosphate group, and/or altered polymerase releasing the cleavage product.
6. The method of claim 1 , wherein the energy transfer donor moiety is a nanoparticle or a fluorescent dye.
7. The method of claim 6 , wherein the nanoparticle is an inorganic fluorescent nanoparticle.
8. The method of claim 6 , wherein the nanoparticle is 1-20 nm in its largest dimension.
9. The method of claim 6 , wherein the nanoparticle is a non-blinking nanoparticle.
10. The method of claim 1 , wherein the target nucleic acid molecule is immobilized to a surface.
11. The method of claim 1 , wherein the at least one type of nucleotide comprises 3-10 phosphate groups.
12. The method of claim 1 , wherein a terminal phosphate group of the at least one type of nucleotide is linked to the acceptor moiety.
13. The method of claim 1 , wherein the polymerase is contacted with at least two types of nucleotide and the types of nucleotides is selected from a group consisting of adenosine, guanosine, cytosine, thymidine and uridine.
14. The method of claim 13 , wherein the at least two types of nucleotides are each linked to a different type of energy transfer acceptor moiety.
15. The method of claim 14 , wherein the energy transfer acceptor moiety is a fluorescent dye.
16. The method of claim 2 , wherein the excitation source is electromagnetic energy.
17. The method of claim 2 , wherein the excitation source is light.
18. The method of claim 2 , wherein the energy transfer signal is a FRET signal.
19. The method of claim 2 , wherein the energy transfer signal is optically detectable.