FLOWCELL SYSTEMS FOR SINGLE MOLECULE DETECTION

• US 20110039266A1
• Filed: 08/10/2010
• Published: 02/17/2011
• 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., Williams, John G.K., Chen, Jiyan, Narayanan, Nara, Sheaff, Pamela
• Granted Patent: US 8,148,516 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-48. -48. (canceled)

49. A nucleotide, said nucleotide having a detectable label attached to the terminal phosphate, wherein said detectable label is linked to said terminal phosphate with a linker comprising a peptide of 2 to 15 amino acids in length.

50. The nucleotide according to claim 49, 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.

51. The nucleotide of claim 49, wherein said detectable moiety is a fluorogenic dye.

52. The nucleotide of claim 49, 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.

53. The nucleotide of claim 52, wherein said detectable moiety is selected from the group consisting of coumarin dyes, xanthene dyes, resorufins, cyanine dyes, difluoroboradiazaindacene dyes (BODIPY), ALEXA dyes, indoles, bimanes, isoindoles, dansyl dyes, naphthalimides, phthalimides, xanthenes, lanthanide dyes, rhodamines and fluoresceins.

54. The nucleotide of claim 52, wherein the said detectable moiety is a member selected from the group consisting of 5-carboxyfluorescein (FAM), rhodamine, 5-(2′

-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, Reactive Red 4, BODIPY dyes and cyanine dyes.

55. The nucleotide of claim 49, wherein the sugar moiety of said nucleotide is selected from the group consisting of ribosyl, 2′

-deoxyribosyl and other modified sugars.

56. The nucleotide of claim 49, wherein the nucleobase of said nucleotide 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.

57. The nucleotide of claim 49, which is a substrate for a nucleic acid polymerase.

58. The nucleotide of claim 49, wherein the detectable moiety after removal of phosphates interacts with additional chemical and/or enzymatic moieties to generate a signal.

59. The nucleotide of claim 58, wherein said additional detection reagents are capable of a response that is detectably different from said detectable species.

60. The nucleotide of claim 49, wherein said peptide is 8 amino acids in length.

61. The nucleotide of claim 60, wherein said nucleotide is compound 55:

62. The nucleotide of claim 49, wherein said peptide is 3 amino acids in length.

63. The nucleotide of claim 62, wherein said peptide of 3 amino acids is KKK.

64. The nucleotide of claim 49, wherein said nucleotide has the formula:

65. 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 of claim 49, 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.

66. The method of claim 65, wherein step (a) further includes conducting said polymerase reaction in the presence of a phosphate or polyphosphate transferring enzyme.

67. The method of claim 65, wherein step (a) further includes conducting said polymerase reaction in the presence of two or more terminal-phosphate-labeled nucleotides with distinct fluorophore moieties.

68. The method of claim 65, 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.

69. The method of claim 65, further comprising the step of including one or more additional detection reagents in said polymerase reaction.

70. The method of claim 69, wherein said additional detection reagents are capable of a response that is detectably different from said fluorophore.