Methods for processing and analyzing nucleic acid samples

• US 8,431,340 B2
• Filed: 10/23/2009
• Issued: 04/30/2013
• Est. Priority Date: 09/15/2004
• Status: Active Grant

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Jovanovich, Stevan Bogdan, Blaga, Iuliu Ioan
• Primary Examiner(s): Nguyen, Dave
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US12/605,217
• Publication Number:
  US 20100068723A1
• Time in Patent Office: 1,285 Days
• Field of Search: 435/6.1, 435/6.11, 435/91.2, 435/173.9, 435/287.2, 435/288.6, 204/451, 422/68.1, 536/23.1, 536/24.33, 536/25.4
• US Class Current: 435/6.1
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/028 Modular arrangements
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0806 Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0874 Three dimensional network
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1844 : using fans
• CPC Class Codes: B01L 2400/0409 : centrifugal forces
• CPC Class Codes: B01L 2400/0421 : electrophoretic flow
• CPC Class Codes: B01L 2400/043 : magnetic forces
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0638 : membrane valves, flap valves
• CPC Class Codes: B01L 2400/0655 : pinch valves
• CPC Class Codes: B01L 2400/086 : using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• 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: 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: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 1/34 : Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 27/44721 : by optical means
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C

Abstract

Methods and devices for the interfacing of microchips to various types of modules are disclosed. The technology disclosed can be used as sample preparation and analysis systems for various applications, such as DNA sequencing and genotyping, proteomics, pathogen detection, diagnostics and biodefense.

Claims

1. A method for processing and analyzing a nucleic acid sample comprising the steps of:

a) concentrating a nucleic acid analyte in a macroscale module by;

(i) providing a sample comprising a nucleic acid analyte in an initial volume into an off-chip macroscale module configured to hold a volume of a milliliter to a liter;

(ii) capturing the nucleic acid analyte from said initial volume onto a plurality of magnetic beads in the macroscale module;

(iii) immobilizing the magnetic beads with the captured nucleic acid analyte in the macroscale module with a magnet;

(iv) washing the magnetic beads with the captured nucleic acid analyte; and

(v) releasing the washed beads into a reduced volume compared with said initial volume to concentrate the nucleic acid analyte;

b) introducing, with pressure from a pressure source, the released magnetic beads with the captured nucleic acid analyte in the reduced volume into a bioprocessor module that is fluidically integrated with the macroscale module, wherein introducing comprises passing the magnetic beads with the captured nucleic acid analyte from the macroscale module through a microfluidic channel in a microfluidic chip and into a reaction chamber in the bioprocessor module wherein the microfluidic channel is in fluidic communication with the macroscale module and with the reaction chamber;

c) amplifying the nucleic acid analyte in the reaction chamber; and

d) analyzing the amplified nucleic acid analyte in an analysis module fluidically integrated with the bioprocessor module.

2. The method of claim 1, further comprising eluting the analytes from the magnetic beads in the bioprocessor module.

3. The method of claim 1, wherein the introducing comprises pumping the magnetic beads onto the microfluidic chip using an on-chip diaphragm pump comprising a series of three diaphragm valves.

4. The method of claim 1, wherein the initial volume comprising the analyte in the macroscale module is one milliliter to one liter.

5. The method of claim 1, wherein the macroscale module further comprises a reservoir containing the magnetic beads.

6. The method of claim 5, wherein the microfluidic chip further comprises an on-chip diaphragm pump comprising a series of three diaphragm valves, and the method comprises pumping the beads from the reservoir into the microfluidic chip.

7. The method of claim 1 wherein the sample comprises cells and the method further comprises lysing the cells in the off-chip macroscale module.

8. The method of claim 7 further comprising moving a lysis buffer into the macroscale module that holds the cells.

9. The method of claim 1 wherein the analyzing comprises capillary electrophoresis.

10. The method of claim 1 performed on a plurality of samples.

11. The method of claim 1 wherein the magnetic beads bind DNA.

12. The method of claim 1 wherein the nucleic acid analyte is DNA.

13. The method of claim 1 wherein the nucleic acid analyte is RNA.

14. The method of claim 1 wherein the system further comprises PCR reagents.

15. The method of claim 1 wherein the amplification comprises performing PCR on variable number tandem repeats (VNTR).