MICROFABRICATED INTEGRATED DNA ANALYSIS SYSTEM

• US 20120142010A1
• Filed: 02/13/2012
• Published: 06/07/2012
• 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., LIU, Chung, KUMARESAN, Palani, BLAZEJ, Robert, YEUNG, Stephanie H.I.
• Granted Patent: US 8,420,318 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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 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.

2. The process of claim 1 wherein the microreactor element includes a microcarrier element which carries the multiple copies of the clonal sequencing template.

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

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

5. The process of claim 1 wherein the distributing step is done such that only one microreactor element will pass into one thermal cycling chamber.

6. The process of claim 5 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.

7. The process of claim 1 wherein the generating step comprises generating multiple clonal copies of a sequencing template by emulsion PCR reactions or by a flow through PCR process.

8. The process of claim 1 wherein the selecting step is fluorescence activated cell sorting (FACS).

9. 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.

10. The process of claim 9 wherein the capturing step uses an oligonucleotide capture matrix.

11. 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.

12. 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.

13. The method of claim 12 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 microsphere out of the constriction and into a main body portion of the thermal cycling chamber before thermal cycling.