Nucleic acid sequencing using charge-switch nucleotides

• US 6,869,764 B2
• Filed: 06/06/2001
• Issued: 03/22/2005
• Est. Priority Date: 06/07/2000
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: L
• Inventors: Bashford, Gregory R., Williams, John G. K.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/876,375
• Publication Number:
  US 20020039738A1
• Time in Patent Office: 1,385 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.37
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation without interference from unincorporated NPs and without illuminating the polymerase-DNA complex.

Claims

1. A method for separating an intact NP probe from a phosphate detectable moiety, said method comprising:

a) providing a sample comprising an intact NP probe with a detectable moiety attached thereto, whereupon an enzymatic cleavage of said intact NP probe to incorporate said NP probe on a primer strand hybridized to a target nucleic acid, a phosphate detectable moiety is produced, wherein said phosphate detectable moiety carries a molecular charge which is different than the molecular charge of said intact NP probe, wherein said intact NP probe is a charge-switch nucleotide phosphate probe having a detectable moiety on a terminal phosphate; and

b) applying an energy field to said sample, thereby separating said phosphate detectable moiety from said sample having an intact NP probe.

2. The method according to claim 1, wherein said charge-switch nucleotide phosphate is a nucleotide triphosphate (NTP) having a γ

-phosphate with a detectable moiety attached thereto.

3. The method according to claim 2, wherein said γ

-phosphate with a detectable moiety attached thereto is a γ

-phosphate with a fluorophore attached thereto.

4. The method according to claim 1, wherein said intact NP probe is incorporated on a primer strand hybridized to a target nucleic acid using a polymerase, thereby releasing said phosphate detectable moiety.

5. The method according to claim 4, wherein said polymerase is immobilized.

6. The method according to claim 1, wherein said energy field is an electric field.

7. The method according to claim 6, wherein said electric field is a first electric field applied in a transverse direction and a second energy field is applied in an axial direction.

8. The method according to claim 7, wherein said second energy field applied in said axial direction is a pressure field.

9. The method according to claim 1, wherein the charge of said phosphate detectable moiety is greater than said intact NP probe.

10. The method according to claim 1, wherein the charge of said phosphate detectable moiety is less than said intact NP probe.

11. The method according to claim 1, wherein the charge of said phosphate detectable moiety is opposite in sign compared to said intact NP probe.

12. The method according to claim 1, further comprising c) detecting said phosphate detectable moiety.

13. The method according to claim 12, wherein said detection is via a charge coupled device (CCD) camera.

14. The method according to claim 12, wherein said detection is via a photodiode.

15. The method according to claim 12, wherein said detection is via a blockade current.

16. A method for identifying an intact charge-switch nucleotide phosphate (NP) probe, said method comprising:

a) contacting a sample comprising said intact charge-switch NP probe having a charged moiety on the base, with an enzyme to produce a phosphate detectable moiety; and

b) applying an electric field to said sample, wherein said phosphate detectable moiety migrates to an electrode differently than said intact charge-switch NP probe.

17. The method according to claim 16, wherein said electrode is an anode.

18. The method according to claim 16, wherein said electrode is a cathode.

19. The method according to claim 16, wherein either said intact NP probe has a positive molecular charge, or wherein upon cleavage of said phosphate detectable moiety, said phosphate detectable moiety carries a positive charge relative to said intact NP probe.

20. The method according to claim 16, wherein said enzyme is selected from the group consisting of a DNA polymerase, a DNA dependent RNA polymerase, a reverse transcriptase, a phosphodiesterase and a phosphatase.

21. The method according to claim 16, wherein said intact charge-switch NP probe is a member selected from the group consisting of a nucleotide diphosphate, a deoxynucleotide triphosphate (dNTP), and a nucleotide triphosphate (NTP).

22. The method according to claim 21, wherein said deoxynucleotide triphosphate (dNTP) is a member selected from the group consisting of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate deoxythymidine triphosphate and deoxyuridine triphosphate.

23. The method according to claim 16, wherein said phosphate detectable moiety is a pyrophosphate with a fluorophore moiety attached thereto.

24. The method according to claim 23, wherein upon cleavage of said pyrophosphate fluorophore moiety, said pyrophosphate fluorophore moiety carries a positive charge relative to said intact NTP probe.

25. The method according to claim 16, wherein said intact NP probe has a positive charge.

26. The method according to claim 16, wherein said intact NP probe has a negative charge.

27. A method for sequencing a nucleic acid, said method comprising:

providing a target nucleic acid, a polymerase priming moiety, a polymerase, and a plurality of intact NP probes;

mixing said target nucleic acid, said polymerase priming moiety, said polymerase and said plurality of NP probes under conditions permitting target dependent polymerization of said plurality of NP probes, such conditions which are capable of providing a time sequence of a plurality of phosphate detectable moieties, wherein said phosphate detectable moieties are used in a sequencing method consisting of one-color sequencing, two-color sequencing, three-color sequencing, four-color sequencing and combinations thereof; and

detecting over time said plurality of phosphate detectable moieties to provide a sequence of said target nucleic acid.

28. The method according to claim 27, wherein said primer moiety is a hairpin loop.

29. The method according to claim 27, wherein said plurality of phosphate detectable moieties independently selected from the group consisting of PPi-Dye, a terminal phosphate fluorophore moiety, a detectable moiety, charged groups, electrically active groups, reporter groups, and combinations thereof.

30. The method according to claim 27, wherein said polymerase is immobilized in single molecule configuration.

31. The method according to claim 27, wherein said phosphate detectable moieties are used in a sequencing method using four-color sequencing.

32. The method according to claim 27, wherein detection occurs as said polymerase incorporates the NP probes in a single nucleic acid molecule.