Chip-based sequencing nucleic acids

• US 8,815,576 B2
• Filed: 12/27/2007
• Issued: 08/26/2014
• Est. Priority Date: 12/27/2007
• Status: Active Grant

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Inventors: Beer, Neil Reginald
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US11/965,585
• Publication Number:
  US 20100184020A1
• Time in Patent Office: 2,434 Days
• Field of Search: 435/6, 435/6.1, 435/6.11, 435/283.1, 435/287.1, 435/287.2, 435/518
• US Class Current: 435/287.2
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• 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 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

Claims

1. An apparatus for sequencing nucleic acids in a sample, wherein the sample contains the nucleic acids;

comprising;

a microchip;

a flow channel in said microchip;

a source of emulsion carrier fluid connected to said flow channel producing an emulsion carrier fluid in said flow channel;

a sample and fluid source that provides a source of the sample that contains the nucleic acids and a fluid;

a sample and fluid channel connected to said sample and fluid source;

a source of magnetic particles;

a magnetic particles channel connected to said source of magnetic particles;

a sample, fluid, and magnetic particles channel connected to said sample and fluid channel and to said magnetic particles channel;

said sample, fluid, and magnetic particles channel connecting the sample containing the nucleic acids, said fluid, and said magnetic particles to said flow channel;

an emulsion microreactor maker connected to said flow channel for producing microreactors containing the sample containing nucleic acids, said fluid, and said magnetic particles,

said emulsion microreactor maker including said emulsion carrier fluid, the sample, said fluid, said magnetic particles, and a junction in said flow channel,

wherein the sample, said fluid, and said magnetic particles are injected into said emulsion carrier fluid in said flow channel producing said microreactors containing the sample, said fluid, and said magnetic particles suspended in said emulsion carrier fluid;

a reagent source connected to said flow channel;

a nucleotides source connected to said flow channel for introducing NTP1 nucleotides, NTP2 nucleotides, NTP3 nucleotides, and NTP4 nucleotides into said flow channel;

a PCR and sequencing zone in said flow channel;

an electromagnet trap for selectively magnetically trapping the nucleic acids and said magnetic particles at said PCR and sequencing zone in said flow channel;

thermalcycler connected to said PCR and sequencing zone in said flow channel; and

a detector for detection and sequencing of the nucleic acids.

2. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said emulsion microreactor maker is a droplet maker for producing droplets that are microreactors containing the nucleic acids and said magnetic particles.

3. The apparatus for sequencing nucleic acid in a sample of claim 1 including magnetic nanoparticles or magnetic polystyrene-coated beads and wherein said flow channel in said microchip has a cross section aspect ratio, width and depth, that is sized to prevent said magnetic nanoparticles or magnetic polystyrene-coated beads from vertical stacking.

4. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said electromagnet trap is in a microfluidic network of parallel or branched microchannels.

5. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said source of emulsion carrier fluid is a source of oil emulsion carrier fluid connected to said flow channel.

6. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said source of emulsion carrier fluid is a source of an electrically insulating, stable fluorocarbon-based fluid emulsion carrier fluid consisting of perfluorohexane or tetradecafluorohexane connected to said flow channel.

7. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said magnetic particles are magnetic-cored optically discrete nanoparticles.

8. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said magnetic particles are magnetic polystyrene-coated beads.

9. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said thermalcycler includes a resistance heater.

10. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said thermalcycler includes a resistive tape heater.

11. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said thermalcycler includes a laser heater.

12. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said detector includes a photodiode.

13. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said detector includes a CCD or CMOS array.

14. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said detector includes a laser.

15. The apparatus for sequencing nucleic acid in a sample of claim 1 wherein said detector includes a LED light source.

16. The apparatus for sequencing nucleic acid in a sample of claim 1 including a cover on said microchip, said cover extending over said

flow channel in said microchip;

said PCR and sequencing zone in said flow channel;

said electromagnet trap for selectively magnetically trapping the nucleic acids and said magnetic particles at said PCR and sequencing zone in said flow channel; and

said thermalcycler connected to said PCR and sequencing zone in said flow channel.

17. An apparatus for sequencing nucleic acid in a sample wherein the sample contains the nucleic acids;

comprising;

a microchip;

a flow channel in said microchip;

a source of emulsion carrier fluid connected to said flow channel producing an emulsion carrier fluid in said flow channel;

a sample and fluid source that provides a source of the sample that contains the nucleic acids and a fluid;

a sample and fluid channel connected to said sample and fluid source;

a source of magnetic particles;

a magnetic particles channel connected to said source of magnetic particles;

a sample, fluid, and magnetic particles channel connected to said sample and fluid channel and to said magnetic particles channel;

said sample, fluid, and magnetic particles channel connecting the sample containing the nucleic acids, said fluid, and said magnetic particles to said flow channel;

an emulsion microreactor maker connected to said flow channel for producing microreactors containing the sample containing nucleic acids, said fluid, and said magnetic particles,

said emulsion microreactor maker including said emulsion carrier fluid, the sample, said fluid, said magnetic particles, and a junction in said flow channel,

wherein the sample, said fluid, and said magnetic particles are injected into said emulsion carrier fluid in said flow channel producing said microreactors containing the sample, said fluid, and said magnetic particles suspended in said emulsion carrier fluid;

a reagent source connected to said flow channel;

a first nucleotides dNTP1 source;

a first nucleotides dNTP1 channel connected to said first nucleotides dNTP1 source and said flow channel for introducing NTP1 nucleotides into said flow channel,

a second nucleotides dNTP2 source;

a second nucleotides dNTP2 channel connected to said second nucleotides dNTP2 source and said flow channel for introducing NTP2 nucleotides into said flow channel,

a third nucleotides dNTP3 source;

a third nucleotides dNTP3 channel connected to said third nucleotides dNTP3 source and said flow channel for introducing NTP3 nucleotides into said flow channel,

a fourth nucleotides dNTP4 source;

a fourth nucleotides dNTP4 channel connected to said fourth nucleotides dNTP4 source and said flow channel for introducing NTP4 nucleotides into said flow channel,

a PCR and sequencing zone in said flow channel;

an electromagnet trap for selectively magnetically trapping the nucleic acids and said magnetic particles at said PCR and sequencing zone in said flow channel;

thermalcycler connected to said PCR and sequencing zone in said flow channel; and

a detector for detection and sequencing of the nucleic acids.

18. An apparatus for sequencing nucleic acids in a sample, wherein the sample contains the nucleic acids;

comprising;

a microchip,

a flow channel in said microchip,

a source of emulsion carrier fluid connected to said flow channel producing an emulsion carrier fluid in said flow channel,

a sample and fluid source that provides a source of the sample that contains the nucleic acids and a fluid;

a sample and fluid channel connected to said sample and fluid source;

a source of magnetic particles;

a magnetic particles channel connected to said source of magnetic particles;

a sample, fluid, and magnetic particles channel connected to said sample and fluid channel and to said magnetic particles channel;

said sample, fluid, and magnetic particles channel connecting the sample containing the nucleic acids, said fluid, and said magnetic particles to said flow channel;

an emulsion droplet maker connected to said flow channel for producing droplets containing sample containing the nucleic acids, said fluid, and said magnetic particles,

said emulsion microreactor maker including said emulsion carrier fluid, the sample, said fluid, said magnetic particles, and a junction in said flow channel connecting said sample, fluid, and magnetic particles flow channel and said flow channel,

wherein the sample, said fluid, and said magnetic particles are injected into said emulsion carrier fluid in said flow channel producing said droplets containing the sample, said fluid, and said magnetic particles suspended in said emulsion carrier fluid;

a reagent source connected to said flow channel,

a first nucleotides dNTP 1 source connected to said flow channel for introducing NTP1 nucleotides into said flow channel,

a second nucleotides dNTP2 source connected to said flow channel for introducing NTP2 nucleotides into said flow channel,

a third nucleotides dNTP3 source connected to said flow channel for introducing NTP3 nucleotides into said flow channel,

a fourth nucleotides dNTP4 source connected to said flow channel for introducing NTP4 nucleotides into said flow channel,

a PCR and sequencing zone in said flow channel,

an electromagnet trap for selectively magnetically trapping the nucleic acids and said magnetic particles at said PCR and sequencing zone in said flow channel,

thermalcycler connected to said PCR and sequencing zone in said flow channel, and

a detector for detection and sequencing of the nucleic acids.

19. The apparatus for sequencing nucleic acid in a sample of claim 18 including a cover on said microchip, said cover extending over said flow channel in said microchip;

said PCR and sequencing zone in said flow channel;

said electromagnet trap for selectively magnetically trapping the nucleic acids and said magnetic particles at said PCR and sequencing zone in said flow channel; and

said thermalcycler connected to said PCR and sequencing zone in said flow channel.

20. The apparatus for sequencing nucleic acid in a sample of claim 19 wherein said cover includes at least one window for said detector for detection and sequencing of the nucleic acids.