Apparatus for loading molecules onto substrates

• US 9,381,517 B2
• Filed: 01/07/2014
• Issued: 07/05/2016
• Est. Priority Date: 03/23/2011
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Pham, Thang, Gray, Jeremy, Fehr, Adrian, Chmura, AJ, Tsai, Yu-Chih, Bhamidipati, Arunashree
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/149,402
• Publication Number:
  US 20140193315A1
• Time in Patent Office: 910 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 9/523 for multisample carriers, e.g. used for microtitration plates
• 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 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

Disclosed are compositions, devices, and methods for loading molecules of interest onto a substrate by contacting beads having molecules of interest attached to them with the substrate, for example by providing a field that brings the beads into proximity or contact with the substrate and moves the beads with respect to the substrate. Such molecules of interest can be deposited onto substrates for single-molecule analysis.

Claims

1. An apparatus comprising:

a fixture for holding a substrate;

a substrate held within the fixture comprising an array of zero mode waveguides on its top surface,

a reservoir for containing a solution in contact with the top surface of the substrate;

a solution, in contact with the substrate, comprising magnetic beads having molecules-of-interest attached thereto, the magnetic beads having diameters greater than the smallest cross-sectional dimensions of the zero mode waveguides; and

a device for generating a dynamic magnetic field disposed below, adjacent to, or above the substrate capable of generating a magnetic field that (i) pulls the magnetic beads to the top surface of the substrate, and (ii) moves the magnetic beads across the top surface of the substrate, whereby molecules-of-interest are deposited within the zero mode waveguides on the substrate.

2. The apparatus of claim 1 wherein the molecules-of-interest comprise template nucleic acids and are bound to the magnetic bead via hybridization between an oligonucleotide attached to the magnetic bead and a sequence on the template nucleic acid.

3. The apparatus of claim 1 wherein the molecules-of-interest comprise polymerase-template nucleic acid complexes.

4. The apparatus of claim 3 wherein the magnetic bead is attached to a hook oligonucleotide comprising a retrieval sequence that is complimentary to an oligonucleotide attached to the magnetic bead and a capture sequence that is complementary to the template nucleic acid.

5. The apparatus of claim 4 wherein the oligonucleotide attached to the magnetic bead comprises a poly(dA), poly(A), poly(dT) or poly(T) sequence.

6. The apparatus of claim 4 wherein the relative binding strength of each of i) the magnetic bead to the hook oligonucleotide, ii) the hook oligonucleotide to the template nucleic acid, and iii) the polymerase-template nucleic acid complex to the substrate are controlled such that when the polymerase-nucleic acid complex becomes bound to the substrate while applying the dynamic magnetic field, an attachment between the hook oligonucleotide and the template nucleic acid is broken.

7. The apparatus of claim 1 wherein the dynamic magnetic field is produced using one or more moving permanent magnets.

8. The apparatus of claim 7 wherein the moving permanent magnets comprise button magnets, bar magnets, or sheet magnets.

9. The apparatus of claim 7 wherein the movement of the magnets is substantially circular.

10. The apparatus of claim 1 wherein the dynamic field is produced using one or more electromagnets.

11. The apparatus of claim 1 wherein the zero mode waveguides are cylindrical.

12. The apparatus of claim 1 wherein the bases of the zero mode waveguides comprise a coupling agent.

13. The apparatus of claim 12 wherein the coupling agent comprises biotin.

14. The apparatus of claim 13 wherein the molecules-of-interest comprise polymerase-nucleic acid complexes, and the polymerase-nucleic acid complexes are attached to a biotin binding protein which binds to the coupling agent.

15. The apparatus of claim 14 wherein the biotin binding protein comprise streptavidin, neutravidin, or avidin.

16. The apparatus of claim 1 wherein the diameter of the bead is 2 times greater or more than the smallest cross-sectional dimension of the zero mode waveguide.

17. The apparatus of claim 1 wherein the diameter of the bead is 2 times greater to 10,000 times greater than the smallest cross-sectional dimension of the zero mode waveguide.

18. The apparatus of claim 1 wherein the zero mode waveguides are cylindrical, and the smallest cross sectional dimensions are the diameters of the zero mode waveguides.

19. The apparatus of claim 1 wherein the magnetic beads are ferromagnetic, paramagnetic, or superparamagnetic.

20. The apparatus of claim 1 wherein the magnetic beads comprise the inorganic materials carbon, iron oxide, silica, or silicon.