Microfabricated integrated DNA analysis system

• US 8,420,318 B2
• Filed: 02/13/2012
• Issued: 04/16/2013
• 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: Mathies, Richard A., Blazej, Robert, Liu, Chung, Yeung, Stephanie H. I., Kumaresan, Palani
• Primary Examiner(s): Kapushoc, Stephen
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US13/372,376
• Publication Number:
  US 20120142010A1
• Time in Patent Office: 428 Days
• Field of Search: 435/6.1, 435/97.2, 435/283.1, 435/287.6, 435/288.6, 204/451, 536/25.4
• US Class Current: 435/6.1
• 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 method comprising:

providing a microfabricated structure comprising a distribution channel, a plurality of second channels, and component separation channels integrated on the same microfabricated structure, wherein each of the second channels is connected to the distribution channel and each of the plurality of second channels comprises a thermal cycling reactor connected to a purification chamber connected to a component separation channel;

distributing a plurality of microcarrier elements, each carrying multiple clonal copies of a sequencing template, from the distribution channel into the thermal cycling reactors of the plurality of second channels such that only one microcarrier element of the plurality of microcarrier elements passes into a particular thermal cycling reactor;

producing thermal cycling extension fragments from the plurality of microcarrier elements in the thermal cycling reactors;

capturing and concentrating the extension fragments in the purification chambers; and

separating the extension fragments from each other based on their relative sizes in the component separation channels.

2. The method of claim 1, further comprising:

prior to distributing the plurality of microcarrier elements into the thermal cycling reactors, performing the following operations;

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 microcarrier elements carrying multiple clonal copies of a sequencing template; and

selecting which microcarrier elements have a sequencing template.

3. The method of claim 2, wherein the generating step comprises generating multiple clonal copies of a sequencing template by emulsion PCR reactions or by a flow through PCR process.

4. The method of claim 2, wherein the selecting step is fluorescence activated cell sorting (FACS).

5. The method of claim 1, further including using an autovalve at an exit port of each thermal cycling chamber to ensure that only one microcarrier element will flow into one thermal cycling chamber.

6. The method of claim 1, wherein capturing the extension fragments comprises using an oligonucleotide capture matrix.

7. The method of claim 1, further comprising:

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

8. The method of claim 7 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 the microcarrier element out of the constriction and into a main body portion of the thermal cycling chamber before thermal cycling.

9. The method of claim 7, wherein the shape of the constriction is substantially circular.

10. The method of claim 7, wherein the shape of the constriction is substantially semicircular.

11. The method of claim 1, further comprising using a timing mechanism and valving together to permit one of the plurality of microcarrier elements flow into a thermal cycling reactor that does not already contain a microcarrier element and to prevent any of the plurality of microcarrier elements from flowing into any thermal cycling reactor that already contains one of the plurality of microcarrier elements.

12. The method of claim 1, wherein each of the plurality of microcarrier elements comprises a microsphere carrying the multiple copies of the sequencing template.

13. The method of claim 12, wherein the diameter of the microsphere is between about 1 and 100 microns.

14. The method of claim 13, wherein the diameter of the microsphere is about 10 microns.

15. The method of claim 1, wherein each component separation channel comprises a capillary electrophoresis channel.

16. The method of claim 1, further comprising using at least one of an optical detector and a timing mechanism to ensure only one microcarrier element passes into a thermal cycling reactor.

17. The method of claim 16, wherein the optical detector comprises an optical scanner that detects light from a microcarrier element.

18. The method of claim 1, wherein the microfluidic structure comprises a plurality of valves and an elastomer membrane, and further comprising applying pneumatic pressure or vacuum on the membrane to actuate the valves.