Large scale DNA microsequencing device

• US 5,944,971 A
• Filed: 04/23/1997
• Issued: 08/31/1999
• Est. Priority Date: 09/29/1995
• Status: Expired due to Fees

• Current Assignee: Lockheed Martin Energy Research Corporation (Martin Marietta Corporation)
• Original Assignee: Lockheed Martin Energy Research Corporation (Martin Marietta Corporation)
• Inventors: Foote, Robert S.
• Primary Examiner(s): Warden, Robert
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/838,962
• Time in Patent Office: 860 Days
• Field of Search: 204/606, 204/607, 204/608, 204/609, 204/610, 204/612, 204/613, 204/614, 204/615, 204/616, 204/617, 204/618, 204/619, 204/620, 204/621, 204/456, 204/457, 204/461, 204/465 , 204/466, 204/467, 204/468, 204/469, 204/470, 435/287.2, 435/6
• US Class Current: 204/456
• 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. A method for sequencing polynucleic acids comprising the steps of:

a) attaching at least one DNA probe capable of acting as a DNA synthesis primer to a surface of a reaction chamber juxtaposed at least one gel electrophoresis lane, said DNA probe comprising a linker covalently attached to said surface of said reaction chamber and a linker oligonucleotide strand covalently attached to said linker;

b) introducing into said reaction chamber a target DNA containing a sequence complementary to said DNA probe, and thereby allowing said DNA probe and said target DNA to hybridize;

c) introducing into said reaction chamber reagents, labeling means and enzymes for dideoxy sequencing, and allowing sequencing reactions to form labelled terminated chains;

d) separating said labelled terminated chains from said target DNA; and

e) applying a voltage across said at least one gel electrophoresis lane to electrophoretically separate said labelled terminated chains.

2. The method according to claim 1, wherein said DNA probe further comprises a primer strand chemically attached to each linker oligonucleotide strand.

3. The method according to claim 2, wherein said linker oligonucleotide strand extends from said linker in a 3'"'"' to 5'"'"' orientation.

4. The method according to claim 3, wherein forming said complementary polynucleotide strand further comprises delivering a group of probe/primers to said linker of said reaction chamber, allowing said linker oligonucleotide strand of said reaction chamber to select one species of probe/primer from said group of probe/primers, said selection accomplished by anti-parallel complementary base pairing with said 5'"'"' end portion of said linker oligonucleotide strand to said probe/primer.

5. The method according to claim 2, further comprising placing a plurality of DNA probes respectively in a corresponding plurality of reaction chambers, each juxtaposed a corresponding gel electrophoresis lane, delivering a plurality of polynucleic acid target strands to each reaction chamber, allowing said primer strand to hybridize with a species of said plurality of polynucleic acid target strands, thereby forming a plurality of hybridized strand species, one said hybridized strand species in each of said reaction chambers, said species of said plural nucleic acid strands being determined by complementary base pairing through hydrogen bonding.

6. The method according to claim 5, further comprising adding labeling means to said hybridized strands to produce plural sequencing strand species for a sequence determining reaction.

7. The method according to claim 1, wherein said sequencing reaction is a Sanger sequencing reaction.

8. The method according to claim 1, wherein said linker is a photocleavable linker.

9. A method for sequencing polynucleic acids comprising the steps of:

a) attaching at least one DNA probe capable of selecting a target DNA sequence to a surface of a reaction chamber juxtaposed at least one gel electrophoresis lane, said DNA probe comprising a linker covalently attached to said surface of said reaction chamber and a linker oligonucleotide strand covalently attached to said linker;

b) introducing into said reaction chamber a target DNA containing a sequence complementary to said DNA probe, and thereby allowing said DNA probe and said target DNA to hybridize;

c) introducing into said reaction chamber reagents, and labeling means for chemical degradation sequencing, and allowing sequencing reactions to form shortened sequencing chains;

d) separating said shortened sequencing chains from said probe DNA; and

,

e) applying a voltage across said at least one gel electrophoresis lane to electrophoretically separate said shortened sequencing chains.

10. The method according to claim 9, wherein said sequencing reaction is a Maxam-Gilbert sequencing reaction.

11. The method according to claim 9, wherein said linker is a photocleavable linker.

12. A method of sequencing a target DNA sequence comprising the steps of:

a) attaching a plurality of probe/primers respectively to a surface of corresponding reaction chambers wherein each of said probe/primers is covalently attached to a linker which is covalently attached to said reaction chamber, each probe/primer having a unique and defined sequence which allows said probe/primer to hybridize to a specific sequence, at different locations, in said target DNA and to act as a primer for DNA sequencing reactions;

b) forming a gel channel respectively in communication with corresponding ones of said reaction chambers;

c) adding a solution containing said target DNA sequence to said reaction chambers;

d) adding reagents and enzymes to permit dideoxy sequencing reactions which form chain terminated labelled strands initiated at different locations of said target DNA;

e) separating labelled strands, and

f) analyzing said separated strands for DNA sequence.

13. The method according to claim 12, wherein said linker is a photocleavable linker.