Method for processing polynucleotide-containing samples

• US 8,852,862 B2
• Filed: 11/16/2005
• Issued: 10/07/2014
• Est. Priority Date: 05/03/2004
• Status: Active Grant

• Current Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Original Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Inventors: Wu, Betty, Althaus, John S., Phadke, Nikhil, Brahmasandra, Sundaresh N., Handique, Kalyan, Kehrer, Aaron, Parunak, Gene, Haley, Cecelia, Springer, Ted
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US11/281,247
• Publication Number:
  US 20060166233A1
• Time in Patent Office: 3,247 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 33/30 Micromixers
• 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 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• 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 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0633 with moving parts
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 2400/0694 vents used to stop and induce flow, backpressure valves
• 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 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• 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 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/5082 : Test tubes per se
• CPC Class Codes: B01L 3/523 : with means for closing or opening
• CPC Class Codes: B01L 3/565 : Seals in general F16L
• 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: C12N 15/1006 : by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/101 : by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0019 : Valves using a microdroplet or microbubble as the valve member
• CPC Class Codes: F16K 99/0032 : using phase transition or influencing viscosity
• CPC Class Codes: F16K 99/0034 : Operating means specially adapted for microvalves
• CPC Class Codes: F16K 99/0036 : operated by temperature variations
• CPC Class Codes: F16K 99/0044 : using thermo-electric means

Abstract

Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that retain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under a second set of conditions (e.g., higher temperature and/or more basic pH). The processing region can be used to, for example, concentrate polynucleotides of a sample and/or separate inhibitors of amplification reactions from the polynucleotides. Microfluidic devices with a processing region are disclosed.

Claims

1. A method for separating one or more polynucleotides from a sample containing polymerase chain reaction inhibitors, the method comprising:

contacting a solution of the sample with a plurality of polynucleotide binding particles, wherein the binding particles are configured to preferentially retain the one or more polynucleotides in the sample as compared to polymerase chain reaction inhibitors;

wherein the sample solution has a volume from 0.5 microliters to 3 milliliters;

wherein the plurality of binding particles have a volume less than 5 microliters, and surfaces that comprise a polycationic polyamide configured to bind polynucleotides in preference to polymerase chain reaction inhibitors at a pH of 8.5 or less;

removing the solution containing inhibitors from the plurality of binding particles; and

releasing the one or more polynucleotides from the binding particles into a single volume of liquid wherein the ratio of the volume of sample solution to the volume of liquid into which the polynucleotides are released is between 50;

1 and 1000;

1, wherein the releasing occurs at a pH of 11.4 or greater.

2. The method of claim 1, wherein the one or more polynucleotides has a size of less than 7.5 Mbp.

3. The method of claim 1, wherein contacting the sample with the plurality of polynucleotide binding particles comprises actuating a thermally actuated pressure source to apply a pressure to the sample.

4. The method of claim 3, wherein contacting the sample with the plurality of polynucleotide binding particles comprises opening a thermally actuated valve to place the sample in fluid communication with the binding particles.

5. The method of claim 1, additionally comprising, prior to the releasing step, washing the particles with a volume of wash solution less than 50 microliters.

6. The method of claim 5 wherein the wash solution comprises a detergent.

7. The method of claim 1, wherein the polymerase chain reaction inhibitors comprise at least one of hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds, DNA binding proteins, or a saccharide.

8. The method of claim 1, wherein the polycationic polyamide is polyethyleneimine.

9. The method of claim 8, wherein the polyethyleneimine has a molecular weight in the range 600-800 Da.

10. The method of claim 1, wherein the polycationic polyamide is selected from the group consisting of poly-DL-ornithine, poly-L-lysine, and poly-D-lysine.

11. The method of claim 1, wherein the releasing comprises heating the plurality of binding particles to a temperature of between about 50°

C. and about 100°

C.

12. The method of claim 11, wherein the plurality of binding particles is heated in the presence of a liquid and the temperature is insufficient to boil the liquid in the presence of the plurality of binding particles during heating.

13. The method of claim 11, wherein the temperature is maintained for between about 1 and 10 minutes.

14. The method of claim 1, wherein the method does not comprise centrifugation of the binding particles.

15. The method of claim 1, wherein the time required for completing the contacting, concentrating, and releasing steps is less than 15 minutes.

16. The method of claim 1, wherein the sample has a volume larger than the volume of the polynucleotide binding particles having the one or more polynucleotides bound thereto by a factor of at least about 10.

17. The method of claim 1, wherein the polycationic polyamide is covalently bound to the surfaces of the binding particles.

18. The method of claim 1, wherein the polycationic polyamide is poly-L-lysine or poly-D-lysine and has an average molecular weight of between about 7,500 Da and about 35,000 Da.

19. The method of claim 1, wherein the polycationic polyamide is poly-L-lysine or poly-D-lysine and has a median molecular weight of between about 15,000 Da and about 25,000 Da.

20. The method of claim 1, wherein the binding particles are made of a polymeric material selected from the group consisting of:

polystyrene, latex polymers, polyacrylamide, and polyethylene oxide.

21. The method of claim 20, wherein the polymeric material is modified to provide one or more carboxylic groups and/or one or more amino groups, wherein the groups provide an attachment point for one or more ligands.

22. The method of claim 1, wherein the binding particles have an average diameter of between about 4 microns and about 20 microns.

23. The method of claim 1, wherein the binding particles are present in a density of about 10⁸ particles per milliliter.

24. The method of claim 1, wherein at least some of the binding particles are magnetic.

25. The method of claim 1, wherein the polycationic polyamide is resistant to pronase degradation.

26. The method of claim 1, wherein the contacting takes place in the presence of lysis reagents.

27. The method of claim 26, wherein the contacting in the presence of lysis reagents comprises heating the sample to a temperature more than 50°

C.

28. The method of claim 26, wherein the contacting in the presence of lysis reagents takes place for a period of time between 5 to 15 minutes.