Microfabricated integrated DNA analysis system

• US 20050287572A1
• Filed: 05/25/2005
• Published: 12/29/2005
• Est. Priority Date: 06/01/2004
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Liu, Chung, Kumaresan, Palani, Blazej, Robert, Mathies, Richard A., Yeung, Stephanie H. I.
• Granted Patent: US 7,799,553 B2
• Time in Patent Office: Days
• US Class Current: 435/6.19
• 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 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• 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 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0633 with moving parts
• 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 3/502753 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 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Methods and apparatus for genome analysis are provided. A microfabricated structure including a microfluidic distribution channel is configured to distribute microreactor elements having copies of a sequencing template into a plurality of microfabricated thermal cycling chambers. A microreactor element may include a microcarrier element carrying the multiple copies of the sequencing template. The microcarrier element may comprise a microsphere. An autovalve at an exit port of a thermal cycling chamber, an optical scanner, or a timing arrangement may be used to ensure that only one microsphere will flow into one thermal cycling chamber wherein thermal cycling extension fragments are produced. The extension products are captured, purified, and concentrated in an integrated oligonucleotide gel capture chamber. A microfabricated component separation apparatus is used to analyze the purified extension fragments. The microfabricated structure may be used in a process for performing sequencing and other genetic analysis of DNA or RNA.

Claims

1. A microfabricated structure comprising:

a distribution channel to distribute microreactor elements carrying multiple copies of a clonal sequencing template into a plurality of thermal cycling chambers such that only one microreactor element will pass into one thermal cycling chamber wherein thermal cycling extension fragments are produced from a microreactor element;

purification chambers connected to the thermal cycling chambers to capture and concentrate the extension fragments; and

component separation channels connected to the purification chambers to analyze the extension fragments.

2. The microfabricated structure of claim 1 wherein the microreactor element includes a microcarrier element that carries the multiple copies of the clonal sequencing template.

3. The microfabricated structure of claim 1 wherein the microreactor element is a bolus or a microemulsion droplet.

4. The microfabricated structure of claim 3 wherein the microreactor element includes a microsphere carrying the multiple copies of the clonal sequencing template.

5. The microfabricated structure of claim 1 wherein the sequencing template is a DNA or RNA sequencing template.

6. A system for performing sequencing comprising:

means for shearing DNA or RNA into fragments;

means for ligating the fragments to form a mixture of desired circular and contaminating linear products;

means for selectively removing the contaminating linear products;

means for generating microreactor elements carrying multiple clonal copies of a single sequencing template;

means for selecting which microreactor elements have a sequencing template;

microfluidic distribution means for distributing a selected microreactor element with a sequencing template into a thermal cycling chamber;

means for ensuring that only one microreactor element will flow into one thermal cycling chamber;

extension means, including the thermal cycling chambers, for producing thermal cycling extension fragments from the microreactor elements carrying multiple copies of the sequencing template;

purification chamber means for capturing, purifying and concentrating the extension fragments; and

component separation means for analyzing the extension fragments.

7. The system of claim 6 wherein the microreactor element includes a microcarrier element that carries the multiple copies of the clonal sequencing template.

8. The system of claim 6 wherein the microreactor element is a bolus or a microemulsion droplet.

9. The system of claim 8 wherein the microreactor element includes a microsphere carrying the multiple copies of the clonal sequencing template.

10. A microfabricated structure comprising:

a distribution channel to distribute microspheres carrying multiple copies of a clonal sequencing template into a plurality of thermal cycling chambers;

an autovalve at an exit port of a thermal cycling chamber to ensure that only one microsphere will flow into one thermal cycling chamber wherein thermal cycling extension fragments are produced from a microsphere;

purification chambers connected to the thermal cycling chambers to capture and concentrate the extension fragments; and

component separation channels connected to the purification chambers to analyze the extension fragments.

11. The microfabricated structure of claim 10 wherein the diameter of a microsphere is between about 1 and 100 microns.

12. The microfabricated structure of claim 11 wherein the diameter of a microsphere is about 10 microns.

13. The microfabricated structure of claim 10 wherein each thermal cycling chamber is in fluid communication with a separation channel.

14. The microfabricated structure of claim 10 wherein the sequencing template is a DNA or RNA sequencing template.

15. A microfabricated structure comprising:

microfluidic distribution channel means for distributing a microsphere carrying a sequencing template into a thermal cycling chamber;

autovalving means for ensuring that only one microsphere will flow into one thermal cycling chamber;

extension means, including the thermal cycling chamber, for producing thermal cycling extension fragments from a microsphere carrying a sequencing template;

integrated purification chamber means for capturing, purifying and concentrating the extension fragments; and

component separation means for analyzing the extension fragments.

16. The microfabricated structure of claim 15 wherein the extension means is a Sanger extension means including a plurality of thermal cycling chambers.

17. The microfabricated structure of claim 16 wherein the autovalving means includes an autovalve at an exit port of the thermal cycling chambers.

18. The microfabricated structure of claim 17 wherein the purification chamber means includes purification chambers connected to the thermal cycling chambers.

19. The microfabricated structure of claim 18 wherein the component separation means is a capillary array electrophoresis means including a plurality of microchannels connected to the purification chambers.

20. The microfabricated structure of claim 15 wherein the sequencing template is a DNA or RNA sequencing template.

21. A microfabricated apparatus comprising:

a thermal cycling chamber configured to receive a microsphere carrying a clonal template, the chamber having an inlet port and outlet port wherein the outlet port includes a constriction that is configured to trap a microsphere in the chamber and to substantially block further flow into the thermal cycling chamber.

22. The microfabricated apparatus of claim 21 wherein the shape of the constriction is substantially circular.

23. The microfabricated apparatus of claim 21 wherein the shape of the constriction is substantially semicircular.

24. The microfabricated apparatus of claim 21 wherein a first valve is located in an inlet channel in fluid communication with the inlet port and a second valve is located in an outlet channel in fluid communication with the outlet port.

25. The microfabricated apparatus of claim 21 wherein in operation the second valve is closed before the first valve to move a microsphere out of the constriction and into a main body portion of the thermal cycling chamber before thermal cycling.

26. The microfabricated apparatus of claim 21 further including a purification chamber in fluid communication with the outlet port of the thermal cycling chamber and an outlet port of the purification chamber in fluid communication with a component separation apparatus.

27. A system for performing sequencing comprising:

means for shearing DNA or RNA into fragments;

means for ligating the fragments to form a mixture of desired circular and contaminating linear products;

means for selectively removing the contaminating linear products;

means for generating microspheres carrying multiple clonal copies of a single sequencing template;

means for selecting which microspheres have a sequencing template;

microfluidic distribution channel means for distributing a selected microsphere with a sequencing template into a thermal cycling chamber;

means for ensuring that statistically only one microsphere will flow into one thermal cycling chamber;

extension means, including the thermal cycling chambers, for producing thermal cycling extension fragments from the microspheres carrying multiple copies of the sequencing template;

purification chamber means for capturing, purifying and concentrating the extension fragments; and

component separation means for analyzing the extension fragments.

28. The system of claim 27 wherein the ensuring means is at least one of an autovalve in the thermal cycling chamber, an optical detector, and a timing mechanism.

29. The system of claim 27 wherein the optical detector is an optical scanner that detects light scattered form a microsphere.

30. The system of claim 27 wherein the timing mechanism includes a pneumatic input located adjacent to an inlet of a thermal cycling chamber.

31. A system for performing DNA sequencing comprising:

means for shearing DNA into DNA fragments;

means for ligating the DNA fragments to form a mixture of desired circular and contaminating linear products;

means for exonuclease degradation for selectively removing the contaminating linear products;

emulsion PCR reaction means for generating microspheres carrying multiple clonal copies of a single DNA sequencing template;

fluorescent activated cell sorting (FACS) means for selecting which microspheres have a DNA sequencing template;

microfluidic distribution channel means for distributing a selected microsphere with a DNA sequencing template into a thermal cycling chamber;

autovalving means for ensuring that statistically only one microsphere will flow into one thermal cycling chamber;

Sanger extension means, including the thermal cycling chambers, for producing thermal cycling extension fragments from the microspheres carrying multiple copies of the DNA sequencing template;

integrated purification chamber means for capturing, purifying and concentrating the extension fragments; and

capillary array electrophoresis means for analyzing the extension fragments.

32. A process for performing sequencing comprising:

shearing DNA or RNA into fragments;

ligating the fragments to form a mixture of desired circular and contaminating linear products;

selectively removing the contaminating linear products;

generating microreactor elements carrying multiple clonal copies of a sequencing template;

selecting which microreactor elements have a sequencing template;

distributing the microreactor elements with the sequencing templates into thermal cycling chambers;

producing thermal cycling extension fragments from the microreactor elements carrying multiple copies of the sequencing template;

capturing and concentrating the extension fragments; and

analyzing the thermal cycling extension fragments.

33. The process of claim 32 wherein the microreactor element includes a microcarrier element which carries the multiple copies of the clonal sequencing template.

34. The process of claim 32 wherein the microreactor element is a bolus or a microemulsion droplet.

35. The process of claim 34 wherein the microreactor element includes a microsphere carrying the multiple copies of the clonal sequencing template.

36. The process of claim 32 wherein the distributing step is done such that only one microreactor element will pass into one thermal cycling chamber.

37. The process of claim 36 further including using an autovalve at an exit port of the thermal cycling chambers to ensure that only one microreactor element will flow into one thermal cycling chamber.

38. The process of claim 32 wherein the generating step comprises generating multiple clonal copies of a sequencing template by emulsion PCR reactions or by a flow through PCR process.

39. The process of claim 32 wherein the selecting step is fluorescence activated cell sorting (FACS).

40. A process for performing DNA sequencing comprising:

shearing DNA into DNA fragments;

ligating the DNA fragments to form a mixture of desired circular and contaminating linear products;

selectively removing the contaminating linear products by exonuclease degradation;

generating microspheres carrying multiple clonal copies of a DNA sequencing template by emulsion PCR reactions;

selecting which microspheres have a DNA sequencing template by fluorescence activated cell sorting (FACS);

distributing the microspheres with the DNA sequencing template into thermal cycling chambers;

using an autovalve at an exit port of a thermal cycling chamber to ensure that statistically only one microsphere will flow into one thermal cycling chamber;

producing thermal cycling extension fragments from the microspheres carrying multiple copies of the DNA sequencing template;

capturing, purifying and concentrating the extension fragments; and

analyzing the thermal cycling extension fragments.

41. The process of claim 40 wherein the capturing step uses an oligonucleotide capture matrix.

42. A process for use in sequencing comprising:

receiving a microsphere carrying a clonal template at an inlet port of a thermal cycling chamber; and

using a constriction at an outlet port of the chamber to trap the microsphere in the chamber and to substantially block further flow into the chamber.

43. A method for analysis comprising:

producing a microsphere carrying a sequencing template; and

locating the microsphere in a thermal cycling chamber by use of a constriction at an outlet port in the chamber such that further flow into the chamber is substantially blocked.

44. The method of claim 43 wherein a first valve is located at an inlet port of the thermal cycling chamber and a second valve is located at an outlet port of the thermal cycling chamber such that the second valve may be closed before the first valve to move a mocrosphere out of the constriction and into a main body portion of the thermal cycling chamber before thermal cycling.