Charge-switch nucleotides

• US 6,936,702 B2
• Filed: 06/06/2001
• Issued: 08/30/2005
• Est. Priority Date: 06/07/2000
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Reynolds, Bambi L., Draney, Dan, Bashford, Gregory R., Chen, Jiyan, Narayanan, Nara, Sheaff, Pamela, Williams, John G. K.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/876,374
• Publication Number:
  US 20020042071A1
• Time in Patent Office: 1,546 Days
• Field of Search: 435/6, 435/91.1, 435/183, 435/87, 435/88, 435/89, 436/94, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 536/22.1
• 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 2533/101 Primer extension
• 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 charge-switch nucleotide phosphate (NP) probe, said NP probe comprising:

an intact NP probe having a terminal phosphate with a fluorophore moiety attached thereto via a linker carrying at least one positive charge, wherein said terminal phosphate is a pyrophosphate with a fluorophore moiety attached thereto, said intact NP probe having a first molecular charge associated therewith, whereupon cleavage of said terminal phosphate as a phosphate fluorophore moiety, said phosphate fluorophore moiety carries a second molecular charge, wherein the difference between said first molecular charge and said second molecular charge is at least 0.5.

2. A charge-switch nucleotide phosphate (NP) probe, said NP probe comprising:

an intact NP probe having a terminal phosphate with a fluorophore moiety attached thereto via a linker carrying at least two positive charges, wherein said terminal phosphate is a pyrophosphate with a fluorophore moiety attached thereto, said intact NP probe having a first molecular charge associated therewith, whereupon cleavage of said terminal phosphate as a phosphate fluorophore moiety, said phosphate fluorophore moiety carries a second molecular charge, wherein the difference between said first molecular charge and said second molecular charge is at least 0.5.

3. A charge-switch nucleotide phosphate (NP) probe, said NP probe comprising:

an intact NP probe having a terminal phosphate with a fluorophore moiety attached thereto, said intact NP probe having a first molecular charge associated therewith, whereupon cleavage of said terminal phosphate as a phosphate fluorophore moiety, said phosphate fluorophore moiety carries a second molecular charge, wherein the difference between said first molecular charge and said second molecular charge is at least 0.5, wherein said charge-switch probe is selected from the group consisting of compound 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 in FIGS. 6A-D.

4. An intact charge-switch nucleotide phosphate (NP) probe, wherein upon enzymatic cleavage of said intact charge-switch NP probe to produce a phosphate detectable moiety, said phosphate detectable moiety migrates to an electrode, and said intact charge-switch NP probe migrates to the other electrode, wherein said charge-switch NP probe is a nucleotide triphosphate (NTP) and said phosphate detectable moiety is a pyrophosphate with a fluorophore moiety attached thereto.

5. The intact charge-switch NP probe according to claim 4, wherein upon cleavage of said phosphate detectable moiety as a pyrophosphate fluorophore moiety, said pyrophosphate fluorophore moiety carries a positive charge relative to said intact NTP probe.

6. The intact charge-switch NP probe according to claim 4, wherein upon cleavage of said phosphate detectable moiety as a pyrophosphate fluorophore moiety, said pyrophosphate fluorophore moiety carries a negative charge relative to said intact NTP probe.

7. The intact charge-switch NP probe according to claim 4, wherein said fluorophore moiety is attached to said terminal phosphate via a linker.

8. The intact charge-switch NP probe according to claim 7, wherein said fluorophore linker is an alkylene group having between about 5 to about 12 carbons.

9. The intact charge-switch NP probe according to claim 7, wherein said linker carries at least one positive charge.

10. The intact charge-switch NP probe according to claim 7 wherein said linker carries at least two positive charges.

11. An intact charge-switch nucleotide phosphate (NP) probe, wherein upon enzymatic cleavage of said intact charge-switch NP probe to produce a phosphate detectable moiety, said phosphate detectable moiety migrates to an electrode, and said intact charge-switch NP probe migrates to the other electrode, and wherein said charge-switch NP probe is a member selected from the group consisting of a deoxynucleotide triphosphate (dNTP), and a nucleotide triphosphate (NTP).

12. The intact charge-switch NP probe according to claim 11, wherein said charge-switch NP probe is a deoxynucleotide triphosphate (dNTP).

13. The intact charge-switch NP probe according to claim 12, 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.

14. The intact charge-switch NP probe according to claim 11, wherein said nucleotide triphosphate (NTP) is a member selected from the group consisting of adenosine triphosphate, cytosine triphosphate, guanosine triphosphate and uridine triphosphate.

15. An intact charge-switch nucleotide phosphate (NP) probe, wherein upon enzymatic cleavage of said intact charge-switch NP probe to produce a phosphate detectable moiety, said phosphate detectable moiety migrates to an electrode, and said intact charge-switch NP probe migrates to the other electrode, wherein at least one of the phosphate moieties of said nucleotide phosphate probe has an ionized oxygen atom with a counter-cation associated therewith.

16. The intact charge-switch NP probe according to claim 15, wherein said counter-cation is a metal ion.

17. The intact charge-switch NP probe according to claim 16, wherein said metal ion is selected from the group consisting of Mg⁺⁺, Mn⁺⁺, K⁺ and Na⁺.