Large scale DNA microsequencing device

• US 5,661,028 A
• Filed: 09/29/1995
• Issued: 08/26/1997
• Est. Priority Date: 09/29/1995
• Status: Expired due to Fees

• Current Assignee: Lockheed Martin Energy Systems, Inc. (AES Corporation)
• Original Assignee: Lockheed Martin Energy Systems, Inc. (AES Corporation)
• Inventors: Foote, Robert S.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/536,010
• Time in Patent Office: 697 Days
• Field of Search: 435/6, 435/91.1, 435/270, 435/183, 435/287.2, 435/287.9, 435/288.2, 435/288.4, 435/288.7, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 935/77, 935/78, 935/85, 204/456, 204/450, 422/101, 422/149, 422/50, 422/68.1, 423/324, 216/2, 216/39, 216/56, 216/89
• US Class Current: 435/287.2
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

A microminiature sequencing apparatus and method provide means for simultaneously obtaining sequences of plural polynucleotide strands. The apparatus comprises a microchip into which plural channels have been etched using standard lithographic procedures and chemical wet etching. The channels include a reaction well and a separating section. Enclosing the channels is accomplished by bonding a transparent cover plate over the apparatus. A first oligonucleotide strand is chemically affixed to the apparatus through an alkyl chain. Subsequent nucleotides are selected by complementary base pair bonding. A target nucleotide strand is used to produce a family of labelled sequencing strands in each channel which are separated in the separating section. During or following separation the sequences are determined using appropriate detection means.

Claims

1. An apparatus for performing DNA sequencing, comprising:

a) a body comprising a substrate and a cover plate bonded thereto wherein said coverplate is transparent;

b) a channel pattern formed within said body and including at least one gel channel having a first and second longitudinal ends and being at least partially filled with gel;

c) a reaction chamber in communication with said gel and having attached thereto photocleavable linkers wherein said photocleavable linkers have bound thereto oligonucleotide probes/primers;

d) means for communicating a solution containing at least one target DNA to said reaction chamber for hybridization with said DNA synthesis primers; and

e) means for establishing an electric field between said first and second longitudinal ends of said at least one gel channel, of sufficient strength to impart electrophoretic separation of chain-terminated strands of DNA through said gel.

2. The apparatus of claim 1, wherein said body comprises either silicon, glass, quartz, plastic, or ceramics.

3. The apparatus according to claim 2, wherein the substrate is made of optically transparent material.

4. The apparatus according to claim 1, wherein said channel pattern includes a plurality of gel channels, and said communicating means comprises at least one manifold connecting channel interconnecting said plurality of gel channels at one common end.

5. The apparatus according to claim 1, wherein said means for establishing an electric field comprises first and second electrodes disposed respectively at said first and second opposite ends of said at least one gel channel, and a d.c. power source coupled to said first and second electrodes.

6. The apparatus according to claim 1 wherein said cover plate has access ports for the introduction of substances into said reaction chamber.

7. A method of making a nucleic acid sedquencing microchip apparatus comprising the steps of:

a) forming a channel pattern in a planar surface of a substrate, said channel pattern including at least one gel channel;

b) fixedly attaching a cover plate on said planar surface of said substrate;

c) partially filling said at least one gel channel with a gel, and thereby forming a reaction chamber in the untilled portion wherein said reaction chamber has attached thereto photocleavable linkers wherein said photocleavable linkers have bound thereto oligonucleotide probes/primers; and

d) placing first and second electrodes respectively at opposite ends of said at least one gel channel.

8. The method according to claim 7, wherein said step of partially filling said at least one gel channel includes filling said at least one gel channel with an acrylamide buffer solution, covering said reaction chamber portion of said gel channel with a photo-blocker, photopolymerizing said acrylamide solution within said parallel channels with a light source, whereby acrylamide in said reaction chamber is not polymerized, and then flushing out the remaining acrylamide.

9. The method according to claim 8, wherein said filling step includes electro-osmotically pumping said acrylamide buffer solution into said at least one gel channel.

10. The method according to claim 7, wherein said step of forming a channel pattern includes etching the channel pattern into, said planar surface of said substrate using photolithographic techniques.

11. The method according to claim 7, wherein said step of forming a channel pattern includes micro-machining said channel pattern.

12. The method according to claim 7, wherein said step of forming a channel pattern includes forming a plurality of parallel gel channels, and forming at least one manifold connecting channel that interconnects said plurality of parallel gel channels.

13. An apparatus for simultaneously sequencing a plurality of polynucleic acids comprising:

a) a solid support substrate;

b) a plurality of sequencing channels, each said channel having a reaction well and a separating zone;

c) means for delivering substances to each said reaction well;

d) means for specifically selecting and binding a predetermined target sequence within said reaction well of each said channel;

e) means for individually controlling sequencing reactions in each said reaction well;

f) photocleavable linkers bound to said reaction wells and wherein said photocleavable linkers have bound thereto oligonucleotide probes/primers;

g) means for applying an electrophoretic voltage across separating zone;

h) means for monitoring said electrophoretic separation to determine a sequence of a polynucleic acid from the target sequence in each said reaction well; and

i) a transparent cover plate fixedly attached to said substrate.

14. The apparatus according to claim 13, wherein said solid support substrate is a glass microchip.

15. The apparatus according to claim 13, wherein said plurality of sequencing channels are formed by etching.

16. The apparatus of according to claim 13, wherein said means for delivering substances to each of said reaction wells comprises at least one manifold connecting channel that intersects each of said reaction wells.

17. The apparatus according to claim 16, wherein said means for delivering substances to each said reaction well further comprises an additional manifold connecting channel that intersects each of said reaction wells and being disposed between said reaction well and said separation zone.

18. The apparatus according to claim 16, wherein said means for delivering substances to each said reaction well further comprises electro-osmotic means for pumping reaction compounds through said manifold connecting channel to said reaction well of each said sequencing channel.

19. The apparatus according to claim 13, wherein said means for specifically selecting and binding a predetermined target sequence within said reaction well of each said sequencing channel comprises a linker compound covalently bonded to a primer compound wherein said primer compound is capable of priming a synthetic reaction.

20. The apparatus according to claim 13, wherein said means for specifically selecting and binding a predetermined target sequence within said reaction well of each said sequencing channel comprises a probe/primer compound complementarily hydrogen bonded to a linker compound.

21. The apparatus according to claim 13, wherein said means for applying electrophoretic voltage comprises first and second electrodes disposed at opposite ends of said plurality of sequencing channels.

22. The apparatus according to claim 13, wherein said plurality of sequencing channels number between 50 and 10,000.

23. The apparatus according to claim 13, wherein each sequencing channel is between 10 and 100 μ

m in width.

24. The apparatus according to claim 13, wherein said means for monitoring said electrophoretic separation comprises plural fluorescent labels, each of said fluorescent labels indicating one nucleotide base, where said nucleotide base is an element of the sequence of the polynucleic acid of said reaction well.

25. The apparatus according to claim 13 wherein said cover plate has access ports for the introduction of substances into said reaction wells.