Chip-Based Sequencing Nucleic Acids

• US 20100184020A1
• Filed: 12/27/2007
• Published: 07/22/2010
• 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
• Granted Patent: US 8,815,576 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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, comprising:

a microchip;

a flow channel in said microchip;

a source of carrier fluid connected to said flow channel;

magnetic particles connected to the nucleic acids;

a microreactor maker connected to said flow channel for producing microreactors containing the nucleic acids and said magnetic particles;

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 of claim 1 wherein said microreactor maker is a droplet maker for producing droplets containing the nucleic acids and said magnetic particles.

3. The apparatus for sequencing nucleic acid of claim 1 wherein said microreactor maker is a system for producing liquid slugs that provide microreactors containing the nucleic acids and said magnetic particles.

4. The apparatus for sequencing nucleic acid of claim 1 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.

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

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

7. The apparatus for sequencing nucleic acid of claim 1 wherein said source of carrier fluid is a source of flourinet carrier fluid connected to said flow channel.

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

9. The apparatus for sequencing nucleic acid of claim 1 wherein said magnetic particles are magnetic polystyrene-coated beads.

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

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

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

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

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

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

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

17. The apparatus for sequencing nucleic acid 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.

18. The apparatus for sequencing nucleic acid of claim 17 wherein said cover includes at least one window for said detector for detection and sequencing of the nucleic acids.

19. An apparatus for sequencing nucleic acids, comprising:

a microchip,

a flow channel in said microchip,

a source of carrier fluid connected to said flow channel,

magnetic particles connected to the nucleic acids,

a droplet maker connected to said flow channel for producing droplets containing the nucleic acids and said magnetic particles,

a reagent source connected to said flow channel,

a first nucleotides dNTP1 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.

20. The apparatus for sequencing nucleic acid of claim 19 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.

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

22. A method of sequencing nucleic acids on a microchip, comprising the steps of:

providing a microchannel flow channel in the microchip;

isolating the nucleic acids;

hybridizing the nucleic acids to magnetic nanoparticles or to magnetic polystyrene-coated beads;

forming microreactors in said microchannel flow channel, said microreactors containing the nucleic acids and said magnetic nanoparticles or magnetic polystyrene-coated beads;

positioning said microreactors containing the nucleic acids and said magnetic nanoparticles or magnetic polystyrene-coated beads in a magnetic trap in said microchannel flow channel, and

sequencing the nucleic acids.

23. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of hybridizing the nucleic acids to magnetic nanoparticles or to magnetic polystyrene-coated beads comprises hybridizing the nucleic acids to magnetic nanoparticles.

24. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of hybridizing the nucleic acids to magnetic nanoparticles or to magnetic polystyrene-coated beads comprises hybridizing the nucleic acids to magnetic-cored optically discrete nanoparticles.

25. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of hybridizing the nucleic acids to magnetic nanoparticles or to magnetic polystyrene-coated beads comprises hybridizing the nucleic acids to magnetic polystyrene-coated beads.

26. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of sequencing the nucleic acids comprises pyrosequencing the nucleic acids.

27. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of sequencing the nucleic acids comprises four-color sequencing the nucleic acids by synthesis.

28. The method of sequencing nucleic acids on a microchip of claim 22 wherein the nucleic acids being analyzed include virions, capsids, or individual cells containing the nucleic acids and wherein said step of isolating the nucleic acids comprises lysing the virions, capsids, or individual cells containing the nucleic acids by acoustic wave lysing, or by heating, or by optical heating, or by chemical lysing, or by laser-induced lysing, followed by hybridizing to the magnetic beads.

29. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of positioning said microreactors containing the nucleic acids and said magnetic nanoparticles or magnetic polystyrene-coated beads in a magnetic trap in said microchannel flow channel comprises moving the nucleic acids in a hydrophobic carrier fluid to said magnetic trap.

30. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of positioning said microreactors containing the nucleic acids and said magnetic nanoparticles or magnetic polystyrene-coated beads in a magnetic trap in said microchannel flow channel comprises forcing the nucleic acid into a water-oil emulsification hydrophobic carrier fluid and moving the nucleic acid in said water-oil emulsification hydrophobic carrier fluid to said magnetic trap.

31. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of positioning said microreactors containing the nucleic acids and said magnetic nanoparticles or magnetic polystyrene-coated beads in a magnetic trap in said microchannel flow channel comprises forcing the nucleic acid into liquid slugs in a carrier fluid and moving the nucleic acid in said liquid slugs to said magnetic trap.

32. The method of sequencing nucleic acids on a microchip of claim 22 including the step of flowing reagent over the nucleic acids in said magnetic trap.

33. The method of sequencing nucleic acids on a microchip of claim 22 wherein said step of providing a microchannel flow channel in the microchip comprises providing a microchannel flow channel cross section aspect ratio, width and depth, that prevents said magnetic nanoparticles or magnetic polystyrene-coated beads from vertical stacking.

34. The method of sequencing nucleic acids on a microchip of claim 22 including the step of placing a cover over said microchip during said steps of

isolating the nucleic acids;

hybridizing the nucleic acids to magnetic nanoparticles or to magnetic polystyrene-coated beads;

positioning said microreactors containing the nucleic acids and said magnetic nanoparticles or magnetic polystyrene-coated beads in a magnetic trap in said microchannel flow channel, and

sequencing the nucleic acids.

35. A method of fast DNA sequencing by amplification of genetic material within microreactors, comprising the steps of:

providing a microchannel flow channel,

hybridizing the genetic material to magnetic nanoparticles or to magnetic polystyrene-coated beads,

forming the microreactors in said microchannel flow channel, and

retaining the genetic material in a PCR/Sequencing Zone by a magnetic field.

36. The method of fast DNA sequencing by amplification of genetic material within microreactors of claim 35 wherein the method encounters reaction products or excess reagents, including the step of holding the genetic material in said PCR/Sequencing Zone by said magnetic field and washing away the reaction products or the excess reagents or removing the reaction products or the excess reagents by chemical degradation.

37. The method of fast DNA sequencing by amplification of genetic material within microreactors of claim 35 including the step of turning off the magnetic field permitting the genetic material to be flushed from said PCR/Sequencing Zone to waste or to archival storage.