DNA sequencing
First Claim
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.
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Accused Products
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.
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Citations
8 Claims
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1. Apparatus comprising:
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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. - View Dependent Claims (2, 3, 4)
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5. A method for sequencing DNA comprising the steps of:
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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. - View Dependent Claims (6, 7, 8)
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Specification