Method and apparatus for analysis and sorting of polynucleotides based on size

• US 9,383,337 B2
• Filed: 01/14/2013
• Issued: 07/05/2016
• Est. Priority Date: 09/25/1996
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Volksmuth, Wayne D.
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US13/741,249
• Publication Number:
  US 20130341190A1
• Time in Patent Office: 1,268 Days
• Field of Search: 204601-605, 204451-455, 422/504, 422/505
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0484 Cantilevers
• 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 3/502738 characterised by integrated valves microvalves F16K99/0001
• 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: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10S 366/03 Micromixers: variable geometry from the pathway influences mixing/agitation of non-laminar fluid flow

Abstract

The invention relates to a microfabricated device and methods of using the device for analyzing and sorting polynucleotide molecules by size.

Claims

1. A method of analyzing an individual polynucleotide single-stranded DNA molecule in a solution using a microfluidic device, the method comprising :

transporting the individual molecule through a detection region having a volume of between 1 femtoliter and 1 picoliter that is positioned in a microfluidic channel of the device without hydrodynamically focusing the solution with a sheath fluid, and without previous size separation of polynucleotides in the solution; and

determining the size of the molecule as it passes through the detection region.

2. The method of claim 1 wherein the size of the individual molecule passing through the detection region is calculated and stored in a data storage unit.

3. The method of claim 1 wherein the channel is microfabricated in a silicon substrate.

4. The method of claim 1, wherein the size of the individual molecule is determined by measuring a reporter attached to the DNA molecule as it passes through the detection region.

5. The method of claim 4, wherein the reporter is a fluorescent label.

6. The method of claim 1, wherein the individual molecule is transported through the detection region separate and apart from other polynucleotides that are initially present in the solution.

7. The method of claim 1, further comprising diverting the individual molecule into a branch channel downstream of the detection region if it is determined to have a selected size.

8. The method of claim 1, wherein the microfluidic channel is between about 1 and 10 micrometers in width and about 1 and 10 micrometers in depth.

9. A method of analyzing a plurality of single-stranded DNA molecules using a microfluidic device, the method comprising:

flowing the solution by hydrostatic pressure through a microfluidic channel in the device such that the molecules pass one-by-one through a detection region located within the channel without hydrodynamically focusing the solution with a sheath fluid; and

determining the size of individual molecules in the plurality as they pass through the detection region.

10. The method of claim 9, wherein the concentration of the DNA molecules in the solution has been adjusted so that the probability that the detection region will contain two or more molecules at the same time (P_x2) is no more than 0.1.

11. The method of claim 9, wherein the molecules are attached with a fluorescent label, and the size of each molecules is determined by measuring fluorescence as it passes through the detection region.

12. The method of claim 9, further comprising diverting molecules from the plurality into a branch channel downstream of the detection region when they are determined to have a size within a selected size range.

13. The method of claim 9, wherein the detection region is between 1 femtoliter and 1 picoliter in volume.

14. The method of claim 9, wherein the microfluidic channel is between about 1 and 10 micrometers in width and about 1 and 10 micrometers in depth.