MICROSEQUENCER-WHOLE GENOME SEQUENCER

• US 20090269746A1
• Filed: 04/25/2008
• Published: 10/29/2009
• Est. Priority Date: 04/25/2008
• Status: Abandoned Application

• Current Assignee: Gil Atzmon, Igal Chertkow, Temuri Budagov
• Original Assignee: Gil Atzmon, Igal Chertkow, Temuri Budagov
• Inventors: Atzmon, Gil, Chertkow, Igal, Budagov, Temuri
• Application Number:
  US12/109,934
• Publication Number:
  US 20090269746A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0616 Ball valves
• CPC Class Codes: B01L 2400/0633 with moving parts
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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 3/502738 characterised by integrated valves microvalves F16K99/0001
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The method and apparatus are disclosed to support speedy sequencing of genomes of individuals. The method comprises random digestion of a stretch of DNA; adaptor ligation of adaptor DNA fragments to DNA segments produced in random digestion, each said adaptor DNA fragment containing a sequence which is complementary to a single DNA primer; PCR amplification of the ligated segments produced in adaptor ligation, utilizing a single DNA primer; distributing the ligated segments into one or more pre-defined isolated locations of a sequencing apparatus, each said location containing DNA fragments placed there for capturing a unique kind of digested DNA segments; capturing at each location a unique kind of amplified DNA segments by hybridization with the DNA fragments, dislodging captured DNA segments from DNA fragments; adding DNA sequencing reaction components into the locations; performing sequencing reactions at each location; separating the products of the sequencing reactions in the sequencing apparatus; and determining the sequences of DNA segments captured at individual locations of the sequencing apparatus. The apparatus comprises one or more isolated locations, each location has a reservoir containing DNA fragments placed there for capturing a unique kind of DNA segments from a DNA solution after dispensing the DNA solution into the reservoir; one or more channels performing DNA separation according to size, said channels being associated with one or more reservoirs; one or more gates controlling the flow of substances in the reservoirs; an optical system which induces fluorescence excitation in, and detects fluorescence emission in the channels; and a computer to produce DNA sequence data.

Claims

1. A DNA sequencing method comprising:

performing random digestion of a stretch of DNA;

performing adaptor ligation of adaptor DNA fragments to DNA segments produced in random digestion, each said adaptor DNA fragment containing a sequence which is complementary to a single DNA primer;

performing PCR amplification of the ligated segments produced in adaptor ligation, said PCR amplification utilizing said single DNA primer;

distributing the ligated segments into one or more pre-defined isolated locations of a sequencing apparatus, each said location containing DNA fragments placed thereat for capturing a unique kind of digested DNA segments;

capturing at each said location a unique kind of amplified DNA segments by hybridizing said DNA segments with said DNA fragments;

dislodging captured DNA segments from said DNA fragments at said locations;

adding DNA sequencing reaction components into said locations of the sequencing apparatus;

performing sequencing reactions at said locations of the sequencing apparatus;

performing separation of the products of the sequencing reactions in the sequencing apparatus, DNA dislodged at an individual said location being individually separated; and

determining the sequences of DNA segments captured at individual locations of the sequencing apparatus.

2. The method of claim 1 further comprising assembling the entire sequence of the original stretch of DNA using segment sequences of said stretch.

3. The method of claim 1 further comprising removing unbound DNA from said locations of the sequencing apparatus after capturing the amplified DNA segments.

4. The method of claim 3 further comprising repeating said DNA capturing followed by said unbound DNA removing one or more times.

5. The method of claim 1 wherein said random digestion is performed using restriction enzymes.

6. The method of claim 1 wherein said separation of the products of the sequencing reactions is performed by electrophoresis.

7. The method of claim 1 wherein said DNA capturing, said DNA dislodging, said sequencing reactions, said DNA separation, or said determining of DNA sequences are performed for two or more said locations in parallel.

8. A DNA sequencing apparatus comprising:

means for receiving a DNA solution, said DNA receiving means containing DNA fragments placed therein for capturing a unique kind of DNA segments from said DNA solution, and being capable of supporting a PCR reaction, a DNA sequencing reaction, single stranded DNA hybridization into double stranded DNA, or double stranded DNA strand separation;

means for performing DNA separation according to size, said DNA separation means being associated with said DNA receiving means;

means for controlling the access of DNA into said DNA receiving means and into said DNA separation means;

means for inducing fluorescence excitation in, and for detecting fluorescence emission from DNA separated in said DNA separation means; and

a computer system receiving fluorescence emission information from said fluorescence/emission means and processing said information to produce DNA sequence data.

9. A DNA sequencing apparatus comprising:

one or more isolated locations, each location comprising a reservoir which contains DNA fragments placed therein for capturing a unique kind of DNA segments from a DNA solution after dispensing the DNA solution into said reservoir, said reservoir being capable of supporting a PCR reaction, a DNA sequencing reaction, single stranded DNA hybridization into double stranded DNA, or double stranded DNA strand separation;

one or more channels performing DNA separation according to size, said channels being associated with one or more of said reservoirs;

an optical system which induces fluorescence excitation in, and detects fluorescence emission from separated DNA migrating in said channels; and

a computer system receiving fluorescence emission information from said optical system and processing said information to produce DNA sequence data.

10. The apparatus of claim 9 further comprising one or more gates controlling the access of substances into said reservoirs and into said channels.

11. The apparatus of claim 10 wherein said DNA fragments are attached to a surface of said gates.

12. The apparatus of claim 11 wherein said gates comprise a DNA holding structure which places said DNA fragments into one or more said reservoirs in parallel and opens or closes all the locations at the same time.

16. The apparatus of claim 9 or 10 further comprising a DNA holding structure which places said DNA fragments into one or more said reservoirs.

17. The apparatus of claim 10 wherein said gates comprise a rotating element, said rotating element swiveling between a first position, which allows said DNA segment capture with said DNA fragments in a reservoir, and a second position with allows for a sequencing reaction to proceed in a reservoir.

18. The apparatus of claim 17 wherein said DNA fragments are attached to said rotating element.

19. The apparatus of claim 18 wherein said rotating element in the first position places said DNA fragments into a reservoir and in the second position removes said DNA fragments from the reservoir while closing another reservoir.

20. The apparatus of claim 10 wherein said gates comprise a bending element, said bending element flexing between a first position, which allows said DNA segment capture with said DNA fragments in a reservoir, and a second position with allows for a sequencing reaction to proceed in a reservoir.

21. The apparatus of claim 10 further comprising means for actuating said gates based on electrostatic, electromagnetic, piezoelectricity or thermal principle.

22. The apparatus of claim 10 in which said channels are hollow fiber bundles.

23. The apparatus of claim 10 in which one or more of said channels are defined into one or more substrate plates.

24. The apparatus of claim 23 in which the substrate is glass or silicone or polymers.

25. The apparatus of claim 23 in which a single channel spans more than one substrate plate.

26. The apparatus of claim 24, 25 or 26 in which said channels form curved patterns as to increase their length per unit of substrate area.

27. The apparatus of claim 9 or 10 in which said optical system detects fluorescence emission from more than one channel at the same time while detecting said fluorescence individually for each channel.

13. The apparatus of clam 10 wherein said gates comprise at least one translating element, said translating element moving between a first position, which allows said DNA segment capture with said DNA fragments in a reservoir, and a second position which allows for a sequencing reaction to proceed in a reservoir.

14. The apparatus of claim 13 wherein said translating element has a substantially spherical shape.

15. The apparatus of claim 13 or 14 wherein said DNA fragments are attached to said translating element.