Methods and apparatus for single molecule sequencing using energy transfer detection

• US 8,999,674 B2
• Filed: 07/30/2012
• Issued: 04/07/2015
• Est. Priority Date: 03/27/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Beechem, Joseph, Nikiforov, Theo, Choong, Vi-En, Peng, Xinzhan, Luo, Guobin, Chen, Cheng-Yao, Previte, Michael
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US13/562,159
• Publication Number:
  US 20120329042A1
• Time in Patent Office: 981 Days
• Field of Search: None
• US Class Current: 435/91.1
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

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.

Claims

1. A method for generating an energy transfer signal comprising the steps of:

(a) contacting (i) a modified polymerase 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;

(b) incorporating the nucleotide into the nucleic acid molecule using the polymerase, wherein the incorporating includes (i) forming a nucleotide cleavage product; and

(ii) generating an energy transfer signal through energy transfer between the donor moiety and the acceptor moiety; and

(c) detecting the energy transfer signal, wherein the detecting is not performed in a waveguide, wherein the at least one type of nucleotide includes 4-10 phosphate groups, and wherein and the modified polymerase includes one or more amino acid substitutions which reduce the rate of polymerase dissociation from a nucleotide cleavage product relative to a polymerase lacking the one or more amino acid substitutions, wherein the modified polymerase comprises the amino acid sequence of SEQ ID NO;

1 having histidine at position 370 substituted with glycine, threonine, serine, lysine, arginine, alanine, glutamine, tryptophan, tyrosine, or phenylalanine.

5. 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, wherein the detecting is not performed in a waveguide.

6. The method of claim 1, further comprising the steps of:

a) exciting the energy transfer donor moiety with an excitation source;

b) detecting the energy transfer signal from the energy transfer donor moiety and the energy transfer acceptor moiety which are in close proximity to each other, wherein the detecting is not performed in a waveguide; and

c) identifying the energy transfer signal from the energy transfer accepter moiety.

7. The method of claim 1, wherein the polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or reverse transcriptase.

8. The method of claim 1, wherein the energy transfer donor moiety is a nanoparticle or a fluorescent dye.

9. The method of claim 8, wherein the nanoparticle is an inorganic fluorescent nanoparticle.

10. The method of claim 8, wherein the nanoparticle is 1-20 nm in its largest dimension.

11. The method of claim 8, wherein the nanoparticle is a non-blinking nanoparticle.

12. The method of claim 1, wherein the target nucleic acid molecule is a DNA or RNA molecule.

13. The method of claim 1, wherein the target nucleic acid molecule is immobilized to a surface.

14. The method of claim 1, wherein a terminal phosphate group of the at least one type of nucleotide is linked to the acceptor moiety.

15. The method of claim 1, wherein the at least one type of nucleotide is adenosine, guanosine, cytosine, thymidine or uridine.

16. The method of claim 1, wherein the polymerase is contacted with at least two types of nucleotide.

17. The method of claim 16, wherein the at least two types of nucleotides are each linked to a different type of energy transfer acceptor moiety.

18. The method of claim 17, wherein the energy transfer acceptor moiety is a fluorescent dye.

19. The method of claim 6, wherein the excitation source is electromagnetic energy.

20. The method of claim 6, wherein the excitation source is light.

21. The method of claim 6, wherein the energy transfer signal is a FRET signal.

22. The method of claim 6, wherein the energy transfer signal is optically detectable.

2. A method for generating an energy transfer signal comprising the steps of:

(a) contacting (i) a modified polymerase linked to an energy transfer donor moiety with (ii) a nucleic acid molecule and with (iii) at least one type of a hexaphosphate nucleotide having an energy transfer acceptor moiety;

(b) incorporating the hexaphosphate nucleotide into the nucleic acid molecule using the polymerase, wherein the incorporating includes (i) forming a nucleotide cleavage product; and

(ii) generating an energy transfer signal through energy transfer between the donor moiety and the acceptor moiety; and

(c) detecting the energy transfer signal, wherein the detecting is not performed in a waveguide, wherein and the modified polymerase includes one or more amino acid substitutions which reduce the rate of polymerase dissociation from a nucleotide cleavage product relative to a polymerase lacking the one or more amino acid substitutions, wherein the modified polymerase comprises the amino acid sequence of SEQ ID NO;

1 having histidine at position 370 substituted with glycine, threonine, serine, lysine, arginine, alanine, glutamine, tryptophan, tyrosine, or phenylalanine.

3. A method for generating an energy transfer signal comprising the steps of:

(a) contacting (i) a modified polymerase linked to an energy transfer donor moiety with (ii) a target nucleic acid molecule which is base-paired with a polymerization initiation site having a terminal 3′

OH group and with (iii) at least one type of a nucleotide having an energy transfer acceptor moiety;

(b) incorporating the nucleotide onto the terminal 3′

OH group using the polymerase, wherein the incorporating includes (i) forming a nucleotide cleavage product; and

(ii) generating an energy transfer signal through energy transfer between the donor moiety and the acceptor moiety; and

(c) detecting the energy transfer signal, wherein the detecting is not performed in a waveguide, wherein the at least one type of nucleotide includes 4-10 phosphate groups, and wherein and the modified polymerase includes one or more amino acid substitutions which reduce the rate of polymerase dissociation from a nucleotide cleavage product relative to a polymerase lacking the one or more amino acid substitutions, wherein the modified polymerase comprises the amino acid sequence of SEQ ID NO;

1 having histidine at position 370 substituted with glycine, threonine, serine, lysine, arginine, alanine, glutamine, tryptophan, tyrosine, or phenylalanine.

4. A method for generating an energy transfer signal comprising the steps of:

(a) contacting (i) a modified polymerase linked to an energy transfer donor moiety with (ii) a target nucleic acid molecule which is base-paired with a polymerization initiation site having a terminal 3′

OH group and with (iii) at least one type of a hexaphosphate nucleotide having an energy transfer acceptor moiety;

(b) incorporating the hexaphosphate nucleotide onto the terminal 3′

OH group using the polymerase, wherein the incorporating includes (i) forming a nucleotide cleavage product; and

(ii) generating an energy transfer signal through energy transfer between the donor moiety and the acceptor moiety; and

(c) detecting the energy transfer signal, wherein the detecting is not performed in a waveguide, wherein and the modified polymerase includes one or more amino acid substitutions which reduce the rate of polymerase dissociation from a nucleotide cleavage product relative to a polymerase lacking the one or more amino acid substitutions, wherein the modified polymerase comprises the amino acid sequence of SEQ ID NO;

1 having histidine at position 370 substituted with glycine, threonine, serine, lysine, arginine, alanine, glutamine, tryptophan, tyrosine, or phenylalanine.