DNA sequencing

• US 6,143,151 A
• Filed: 12/11/1995
• Issued: 11/07/2000
• Est. Priority Date: 03/29/1984
• Status: Expired due to Fees

• Current Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Middendorf, Lyle Richard, Brumbaugh, John
• Primary Examiner(s): Woodward, Michael P.
• Assistant Examiner(s): DELACROIX MUIRHEI, CYBILLE
• Application Number:
  US08/570,079
• Time in Patent Office: 1,793 Days
• Field of Search: 935/19, 204/456, 204/461, 204/466, 204/606, 204/612, 204/601, 204/616, 204/451
• US Class Current: 204/451
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding

Abstract

To sequence long strands of DNA, clone strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method of by ethidium bromide.

Claims

1. Apparatus comprising:

means for performing gel electrophoresis;

said means for performing gel electrophoresis having at least four identical separating channels;

each of said at least four identical separating channels including a corresponding first end, second end and a central section;

each of said first ends being adapted to receive DNA fragments terminated at least at one of four nucleic acid molecules, A, G, C, and T;

said means for performing gel electrophoresis further including means for separating bands of the DNA fragments before reaching said second end of said means for performing gel electrophoresis wherein said bands are separated on line at said second end prior to the resolution of the larger molecular weight DNA fragments;

means for identifying four separate groups of DNA fragments in the separated bands in accordance with the terminating base of the DNA fragments;

each of said channels including at least one capillary tube;

said means for performing gel electrophoresis further including means for establishing potential across said at least four identical separate channels, wherein bands of more mobile DNA fragments are fully resolved in the gel electrophoresis channels while some of the less mobile strands to be later formed into bands are unresolved in a continuous process; and

said means for identifying including means for generating electrical signals indicating the time sequence of the marked bands of DNA corresponding to each of the DNA fragments terminated at different ones of the nucleic acid groups.

2. Apparatus according to claim 1 in which said means for identifying includes fluorescent markers and said means for generating electrical signals.

3. Apparatus according to claim 1 in which each of several of said identical separating channels includes at least one capillary tube.

4. Apparatus according to claim 1 in which each of several of said identical separating channels includes a bundle of capillary tubes.

5. A method for sequencing DNA comprising the steps of:

preparing a multiplicity of identical DNA strands;

preparing, from the multiplicity of identical DNA strands, fluorescently marked DNA strands with random lengths terminated at least at one of different ones of the adenine base, guanine base, cytosine base and thymine base, wherein at least one batch of DNA strands is formed, which batch is terminated at one of the adenine base, guanine base, cytosine base and thymine base;

applying samples of the fluorescently marked DNA strands to at least one channel for a separating appartus wherein said at least one channel includes a capillary tube;

separating the strands within at least one channel so that the bands of more mobile strands in the channels are fully resolved while some of the less mobile strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are fully resolved while the less mobile strands are yet unresolved into bands in the channel;

applying light from a source of light to the resolved bands; and

identifying and recording the sequence of the bands in the channel from the reaction of the fluorescently marked strands with the light so as to indicate the DNA sequence.

6. A method according to claim 5 in which the step of preparing marked DNA strands from the multiplicity of identical DNA strands includes the step of terminating the strands during synthesis at different ones of the adenine base, guanine base, cytosine base and thymine base.

7. A method according to claim 5 in which the step of preparing marked DNA strands from the multiplicity of identical DNA strands includes the step of randomly cleaving the DNA strands at least at one of the adenine base, guanine base, cytosine base and thymine base whereby DNA strands are created with random lengths.

8. A method according to claim 5 in which the step of preparing includes the steps of:

fluorescently marking the DNA strands on one end;

dividing the DNA strands into at least four batches; and

cleaving the DNA strands in each of the four batches at different one of adenine, guanine, cytosine and thymine.