Sensing apparatus for amplification and sequencing of template polynucleotides and array for amplification of template polynucleotides

• US 10,316,363 B2
• Filed: 04/24/2017
• Issued: 06/11/2019
• Est. Priority Date: 07/18/2012
• Status: Active Grant

• Current Assignee: DNAE Group Holdings Limited (Genting Bhd.)
• Original Assignee: DNAE Group Holdings Limited (Genting Bhd.)
• Inventors: Ansari, Zahid, Amin, Krishna, Jorgensen, Ginny, Kolb, Kurt, Morley, Daniel, Patel, Alpesh, Reed, Samuel, Shepherd, Leila, Toumazou, Christofer
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/494,832
• Publication Number:
  US 20170226579A1
• Time in Patent Office: 778 Days
• Field of Search: None
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1827 using resistive heater
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Provided is a sensing apparatus comprising a chip for integrated amplification and sequencing of a template polynucleotide in a sample. The apparatus comprises a chip with at least one ISFET in a well or chamber, amplification means for amplifying the template polynucleotide on a surface of said chip and comprising at least one heating means suitable for conducting amplification of the template polynucleotide at temperatures elevated with respect to room temperature, and sequencing means for sequencing the amplified template polynucleotide in said well or chamber. Methods of use are also provided.

Claims

1. A method of operating a sensing apparatus for amplification and sequencing of a plurality of template polynucleotides, wherein the apparatus comprises a microfluidic structure defining a plurality of wells each containing an immobilised template polynucleotide, with each well being exposed to at least one ISFET, the method comprising:

controlling the microfluidic structure to provide a removable seal covering a surface of the microfluidic structure thus substantially isolating each well from adjacent wells; and

amplifying the template polynucleotides in the wells using a heater for elevating a temperature within the wells with respect to room temperature

in order to create a clonal population within each well, and

controlling the microfluidic structure to remove said removable seal; and

sequencing the clonal populations within the same wells in which the amplification occurs whilst using the ISFETs to detect nucleotide insertion, wherein said steps of amplifying and controlling comprise controlling channels and manifolds of the microfluidic structure in order to introduce different nucleotides into the wells for amplification and sequencing.

2. The method according to claim 1, wherein there is at least one oligonucleotide immobilized within each well, which oligonucleotide is unbound to a polynucleotide but adapted to bind to the template polynucleotide.

3. The method according to claim 1, wherein the removable seal is a liquid.

4. The method according to claim 1, wherein the removable seal is a solid.

5. The method according to claim 1, wherein a surface within each well is modified with means to immobilize oligonucleotides.

6. The method according to claim 1, wherein a single oligonucleotide is available in each of said wells to hybridize with the template polynucleotide in the sample.

7. The method according to claim 5, wherein beads provide the surface for immobilization of oligonucleotides.

8. The method according to claim 1, wherein the heater comprises an on-chip heating element and, optionally, also an on-chip temperature sensor and/or on-chip temperature control circuitry.