Absolute PCR Quantification

• US 20150017654A1
• Filed: 07/30/2014
• Published: 01/15/2015
• Est. Priority Date: 10/12/2005
• Status: Abandoned Application

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Gorfinkel, Vera, Gorbovitski, Boris
• Application Number:
  US14/446,654
• Publication Number:
  US 20150017654A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

The present application provides methods and devices for absolute quantification of polymerase chain reaction target nucleic acids. In particular, the methods and devices of the present application provide for splitting a nucleic acid sample to be analyzed into small, isolated volumes, conducting the method of polymerase chain reaction (PCR) on said volumes, detecting PCR amplification products, analyzing said detected PCR amplification products, performing absolute quantification of the PCR target and presenting said quantification results.

Claims

1-57. -57. (canceled)

58. A method of sampling a target nucleic acid, comprising:

a) providing a sample tray comprising a plurality of isolated reaction zones, wherein each isolated reaction zone defines a specific reaction sub-volume, wherein the reaction sub-volumes of said different isolated reaction zones are variable reaction sub-volumes;

b) exposing the sample tray to a sample comprising one or more target nucleic acid molecules under conditions wherein said sample fills said plurality of isolated reaction zones;

c) detecting the target nucleic acid in said isolated reaction zones.

59. The method of claim 58, wherein said variable reaction sub-volumes are serial reaction sub-volumes.

60. The method of claim 59, wherein the serial reaction sub-volumes differ between isolated reaction zones by factors of 2.

64. The method of claim 59, wherein the serial reaction sub-volumes differ between isolated reaction zones by factors of 1000.

66. The method of claim 58, wherein said detecting comprises performing a nucleic acid detection reaction.

67. The method of claim 66, wherein said nucleic acid detection reaction comprises a nucleic acid amplification reaction.

68. The method of claim 67, wherein said nucleic acid amplification reaction is a polymerase chain reaction.

69. The method of claim 68, wherein said detecting is conducted after the completion of said polymerase chain reaction.

70. The method of claim 58, wherein said detecting comprises detection of fluorescence.

71. The method of claim 70, wherein said detecting comprises use of an oligonucleotide probe comprising a fluorescent moiety.

72. The method of claim 71, wherein said oligonucleotide probe further comprises a quencher moiety.

73. The method of claim 70, wherein said detecting comprises use of a nucleic acid intercalating dye.

74. The method of claim 68, wherein said polymerase chain reaction is conducted for a predetermined number of cycles, and wherein said detecting is conducted after completion of said predetermined number of cycles.

61-63. -63. (canceled)

65. (canceled)