HYBRIDISATION COLUMN FOR NUCLEIC ACID ENRICHMENT

• US 20170226501A1
• Filed: 08/14/2015
• Published: 08/10/2017
• Est. Priority Date: 08/14/2014
• Status: Abandoned Application

• Current Assignee: Sysmex Corporation
• Original Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Inventors: MILLER, Oliver, SPEIGHT, Graham, SOUTHERN, Ed, MCANDREW, Mike, HURD, Douglas
• Application Number:
  US15/503,480
• Publication Number:
  US 20170226501A1
• Time in Patent Office: Days
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0663 Whole sensors
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

The invention relates to the rapid enrichment of nucleic acid molecules of interest from complex mixtures of nucleic acids for the purpose of sequencing genes and variants, e.g. for clinical uses as well as other applications. A hybridisation column comprising an inner channel, wherein a portion of said channel is filled completely with a porous solid support comprising (a) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them and the remainder of the channel, and (b) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids is disclosed.

Claims

1-56. -56. (canceled)

57. A hybridisation column comprising an inner channel, wherein a portion of said channel is filled with a porous solid support comprising:

(i) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them and the remainder of the channel, and

(ii) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids,

wherein the pore structure of the solid support is homogeneous in all dimensions, wherein the solid support fills the entire cross-section of the channel.

58. The hybridisation column of claim 57, wherein the voids have an average pore size of 0.1-100 μ

m.

59. The hybridisation column of claim 57, wherein the portion of the channel occupied by the solid support has a volume ranging from 0.1 mm³ to 100 mm³.

60. The hybridisation column of claim 57, wherein the voids within the solid support take up about 30-50% of the volume taken up by the solid support in the channel.

61. The hybridisation column of claim 57, wherein one end of the channel comprises a porous filter, frit or permeable membrane to keep the solid support in the column when suction is applied to said end of the channel or pressure is applied to the opposite end of the channel.

62. A microfluidic device comprising one or more hybridisation column(s) according to claim 57, a temperature control element and a temperature sensor, wherein the temperature control element can be used to control the temperature within the channel of the one or more hybridisation column(s).

63. The microfluidic device of claim 62, further comprising one or more reservoir(s) wherein each reservoir is connected to one end of the channel of the one or more hybridisation column(s), wherein each reservoir can be sealed and pressurised.

64. The microfluidic device of claim 63, wherein the pressure is supplied by a source of compressed gas, which is connected to the one or more reservoir(s) via tubing.

65. The microfluidic device of claim 64, wherein pressure applied to the one or more sealed reservoir(s) pushes a fluid within the channel of the one or more hybridisation columns through the solid support.

66. The microfluidic device of claim 64, further comprising a valve within the tubing that connects the source of compressed gas supply to the one or more reservoir(s), wherein the valve can be opened and closed by a controller, which can control the flow rate of fluid driven through the channel of the one or more hybridisation column(s).

67. The microfluidic device of claim 63, wherein the pressure is supplied by a pump, wherein the pump is connected to an electronic controller, which can be programmed to control the flow rate of fluid driven through the channel of the one or more hybridisation column(s).

68. The microfluidic device of claim 63, further comprising one or more collection tubing attached to the other end of the channel of the one or more hybridisation columns.

69. The microfluidic device of claim 64, wherein one end of the channel of the one or more hybridisation columns is connected to a suction pump, wherein the suction pump is connected to a controller, which can control the flow rate of fluid driven through the channel of the one or more hybridisation column(s) when suction is applied.

70. A microfluidic device adapted for receiving the column of claim 57 in order to produce a device comprising one or more hybridisation column(s), a temperature control element and a temperature sensor, wherein the temperature control element can be used to control the temperature within the channel of the one or more hybridisation column(s).

71. A method of preparing the microfluidic device of claim 62 by inserting a hybridisation column comprising an inner channel, wherein a portion of said channel is filled with a porous solid support comprising:

(i) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them and the remainder of the channel, and

(ii) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids,

wherein the pore structure of the solid support is homogeneous in all dimensions, wherein the solid support fills the entire cross-section of the channel.

80. A method of preparing the hybridisation column of claim 57, wherein the method comprises:

(i) providing a column comprising an inner channel, and

(ii) filling the entire cross-section of the channel with a plurality of microbeads having about the same diameter and having linked to their surfaces a plurality of hybridisation probes, wherein the microbeads form a porous solid support.

81. The method of claim 80, wherein the diameter is 0.5 to 500 μ

m.

82. A kit for preparing the hybridisation columns of claim 57, wherein the kit comprises (i) one or more column(s) comprising a inner channel and (i) a container comprising a plurality of microbeads having about the same diameter and having linked to their surfaces a plurality of hybridisation probes.

72. A method for enriching nucleic acid molecules from a complex mixture of nucleic acids, wherein said method comprises:

(i) providing a porous solid support comprising

(a) a plurality of interconnected, micron-sized voids that permit a fluid to flow between them, and

(b) a plurality of hybridisation probes, which are bound to the surfaces of the solid support forming the voids,

(ii) driving the mixture of nucleic acids through the solid support thereby allowing nucleic acid molecules comprising nucleic acid sequences complementary to the nucleic acid sequences of the hybridisation probes to hybridise to the probes, wherein hybridisation is allowed to proceed for less than 10 hours,

(iii) washing the solid support to remove any nucleic acids that are not hybridised to the probes, and

(iv) eluting the nucleic acid molecules bound to the hybridisation probes from the solid support.

73. The method of claim 72, wherein the individual hybridisation probes for each complementary nucleic acid sequence are randomly distributed on the surface of the solid support.

74. The method of claim 72, wherein the mixture is driven through the solid support at a flow rate of 1-100 μ

l/minute.

75. The method of claim 72, wherein hybridisation takes place at a temperature of 55-65°

C.

76. The method of claim 75, wherein the washing step is performed at a temperature 5-10°

C. below the temperature used for hybridisation.

77. The method of claim 72, wherein elution is achieved by heating the solid support to a temperature of about 90-100°

C.

78. The method of claim 72, wherein the hybridisation probes are 50-250-mer oligonucleotide of the same length and/or the nucleic acid molecules in the mixture have a length of 100-350.

79. The method of 72, wherein the mixture is prepared by obtaining a larger nucleic acid molecule, fragmenting it into nucleic acid fragments, and joining the fragments to adaptor molecules.