Charge switch nucleotides

• US 7,659,070 B2
• Filed: 10/26/2007
• Issued: 02/09/2010
• 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 L., Draney, Dan, Bashford, Gregory R., Chen, Jiyan, Narayanan, Nara, Sheaff, Pamela, Williams, John G. K.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/925,722
• Publication Number:
  US 20080153095A1
• Time in Patent Office: 837 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/24.33, 536/26.6
• 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 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. An analytical method for separating an intact charge-switch nucleotide phosphate (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 enzymatic cleavage of said intact NP probe, which produces a phosphate detectable moiety, said phosphate detectable moiety carries a molecular charge which is different than the molecular charge of said intact NP probe; and

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

2. The method according to claim 1, wherein said NP probe with a detectable moiety is a labeled nucleotide triphosphate (NTP).

3. The method according to claim 2, wherein said labeled nucleotide triphosphate (NTP) having a detectable moiety is a NTP having a γ

-phosphate with a detectable moiety attached thereto.

4. The method according to claim 2, wherein said charged detectable moiety when released comprises a pyrophosphate with a fluorophore moiety attached thereto.

5. The method according to claim 3, wherein said labeled NTP is incorporated into said nucleic acid primer hybridized to said target nucleic acid using said polymerase, thereby releasing said γ

-phosphate with said detectable moiety attached thereto.

6. The method according to claim 5, wherein said target nucleic acid comprises a self-complementary region forming said primer.

7. The method according to claim 1, wherein the charge of said detectable moiety after release is different than said labeled nucleotide phosphate (NP) having a detectable moiety attached thereto.

8. The method according to claim 7, wherein the charge of said detectable moiety is more positive than the unincorporated labeled NP.

9. The method according to claim 7, wherein the charge of said detectable moiety attached thereto is opposite in sign compared to the unincorporated fluorescently labeled NP.

10. The method according to claim 1, further comprising

c) measuring said detectable moiety with a measuring device.

11. The method according to claim 10, wherein said measuring device is selected from the group consisting of a charge coupled device (CCD) camera, a photodiode, a video chip, amp meter, voltage meter, and a dye-impregnated polymeric coating on optical fiber sensor.

12. The method according to claim 11, wherein said detection is via a CCD camera.

13. The method according to claim 11, wherein said detection is via a photodiode.