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

• US 20060035273A1
• Filed: 10/21/2005
• Published: 02/16/2006
• Est. Priority Date: 09/25/1996
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Volksmuth, Wayne D.
• Granted Patent: US 7,670,471 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2015/149 Sorting the particles
• 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 for detecting labeled molecules in a microfluidic device, said method comprising

a) providing labeled molecules, wherein said molecules are labeled with a fluorescent dye;

b) providing a microfluidic device comprising a microfluidic channel, wherein said microfluidic channel comprises a detection region;

c) transporting the labeled molecules through the microfluidic channel such that the molecules pass though the detection region;

d) illuminating labeled molecules in the detection region with radiation from an excitation light source, wherein said radiation is of a wavelength that excites the fluorescent dye, and wherein the fluorescent dye when excited emits light of characteristic wavelengths; and

e) detecting the emitted light,

wherein labeled molecules outside the detection region are not illuminated with radiation from the excitation light source.

2. The method of claim 1 wherein, on average, only one labeled molecule occupies the detection region at a time.

3. A method of sizing polynucleotides in solution, comprising:

a) flowing a continuous stream of solution containing reporter-labeled polynucleotides through a microfabricated channel comprising a detection region having a selected volume, where the concentration of the molecules in the solution is such that most molecules pass through the detection region one by one, and

b) determining the size of each molecule as it passes through the detection