Charge switch nucleotides

• US 7,625,701 B2
• Filed: 06/15/2005
• Issued: 12/01/2009
• Est. Priority Date: 06/07/2000
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Reynolds, Bambi, Bashford, Gregory, Draney, Dan, Chen, Jiyan, Narayanan, Nara, Sheaff, Pamela, Williams, John G. K.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/154,419
• Publication Number:
  US 20060063173A1
• Time in Patent Office: 1,630 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/26.6, 536/25.3
• 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 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 charged-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 compound having Formula I:

2. The compound according to claim 1, wherein said detectable moiety is selected from the group consisting of chemiluminescent compounds, fluorescent or fluorogenic dyes, colored or chromogenic dyes, electrochemical tags and combinations thereof.

3. The compound of claim 1, wherein y is 2.

4. The compound of claim 1, wherein said detectable moiety is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, electrochemical tags and combinations thereof.

5. The compound of claim 1, wherein said detectable moiety is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, reduction/oxidation potential and combinations thereof.

6. The compound of claim 5, wherein said detectable moiety is an enzyme-activatable fluorogenic moiety and is selected from the group consisting of fluorescein diphosphate, fluorescein 3′

(6′

)-O-alkyl-6′

(3′

)-phosphate, 4-methylumbelliferyl phosphate, resorufin phosphate, 4-trifluoromethylumbelliferyl phosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, and 6,8-difluoro-4-methylumbelliferyl phosphate.

7. The compound of claim 5, wherein the said detectable moiety is a fluorescent moiety selected from the group consisting of fluorescein, rhodamine, bodipy, cyanine, Alexa, Naphthofluorescein, Oregon Green, coumarin, dansyl, and Texas Red.

8. The compound of claim 1, wherein said sugar moiety is selected from the group consisting of ribosyl, 2′

-deoxyribosyl and other modified sugars.

9. The compound of claim 1, wherein said nucleobase is selected from the group consisting of uracil, thymine, cytosine, 5-methylcytosine, guanine, 7-deazaguanine, hypoxanthine, 7-deazahypoxanthine, adenine, 7-deazaadenine, 2,6-diaminopurine and analogs thereof.

10. The compound of claim 1, which is a substrate for a nucleic acid polymerase.

11. The compound of claim 1, where the detectable moiety after removal of phosphates interacts with additional chemical and/or enzymatic moieties to generate a signal.

12. The compound of claim 11 , wherein said additional detection reagents are capable of a response that is detectably different from said detectable species.

21. The compound of claim 1, wherein y is 3.

23. The compound of claim 1, wherein X is a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur.

24. The compound of claim 23, wherein X is nitrogen.

13. A method for detecting the presence of a DNA sequence, said method comprising:

a) conducting a DNA polymerase reaction in the presence of a terminal-phosphate-labeled nucleotide, which reaction results in the production of a labeled polyphosphate;

b) permitting said labeled polyphosphate to react with a phosphate or polyphosphate transferring enzyme to produce a fluorophore moiety; and

c) detecting the presence of said fluorophore moiety.

14. The method of claim 13, wherein step (a) further includes conducting said polymerase reaction in the presence of a phosphate or polyphosphate transferring enzyme.

15. The method of claim 13, wherein step (a) further includes conducting said polymerase reaction in the presence of two or more terminal-phosphate-labeled nucleotides with distinct fluorophore moieties.

16. The method of claim 13, wherein said fluorophore moiety is an enzyme-activatable label selected from the group consisting of chemiluminescent compounds, fluorogenic or fluorescent dyes, chromogenic or colored dyes, electrochemical tags and combinations thereof.

17. The method of claim 13, wherein said terminal-phosphate-labeled nucleotide may be represented by Formula I:

18. The method of claim 13, wherein y is 2.

19. The method of claim 13, further comprising the step of including one or more additional detection reagents in said polymerase reaction.

20. The method of claim 19, wherein said additional detection reagents are capable of a response that is detectably different from said fluorophore.

22. The method of claim 17, wherein y is 3.

25. The method of claim 17, wherein X is a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur.

26. The method of claim 25, wherein X is nitrogen.