DNA sequencing
First Claim
1. A method for sequencing a portion of a nucleotide chain from a primary nucleotide to an nth nucleotide, where n is an integer not greater than about 500, said method comprising:
- preparing a first mixture of labeled nucleotide chain fragments, each labeled chain extending from a common end adjacent to said primary nucleotide and extending to a nucleotide at a site ranging from said primary through said nth nucleotide, said first mixture containing labeled fragments of all lengths from that terminating at said primary nucleotide to that terminating at said nth nucleotide;
preparing a second mixture of labeled nucleotide chain fragments, the labeled chain fragments extending from said common end and terminating at each position from said primary nucleotide through said nth nucleotide wherever a first of the four nucleotides occurs and wherever a second of the four nucleotides occurs;
preparing a third mixture of labeled nucleotide chain fragments, the labeled chain fragments extending from said common end and terminating at each position from said primary nucleotide through said nth nucleotide wherever said first nucleotide occurs and wherever a third of the four nucleotides occurs;
electrophoresing said mixtures of fragments on a gel that separates said fragments according to their length, said fragments being applied to said gel to run in side-by-side lanes;
visualizing bands of labeled nucleotide fragments on said lanes of said gel; and
reading the nucleotide sequence according to the band patterns in the direction normal to the lanes.
1 Assignment
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Accused Products
Abstract
A first mixture is prepared that contains labeled chain fragments which each has a common end adjacent to a primary nucleotide and a termination at a position from the primary through an nth nucleotide, the first mixture containing nucleotide chain fragments of each length from termination at the primary through termination of the nth nucleotide. A second mixture is prepared that contains labeled chain fragments beginning at the common end and terminating at positions from the first through the nth nucleotide, the second mixture containing chain fragments of each length terminating wherever either a first or a second of the four nucleotides occurs. A third mixture is prepared that contains labeled chain fragments beginning at the common end and terminating at a position from the first through the nth nucleotide, the third mixture containing chain fragments of each length terminating wherever the first or a third of the four nucleotide sequences occurs. The chains are electrophoresed with the first mixture as the center lane. This three-lane system provides a unique band pattern for each of the four nucleotides and permits the sequence to be read merely by directly comparing each of the flanking lanes with the fully stepped center lane. This system has important advantages in reducing reading errors, particularly when read with computer-assisted scanning apparatus.
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Citations
16 Claims
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1. A method for sequencing a portion of a nucleotide chain from a primary nucleotide to an nth nucleotide, where n is an integer not greater than about 500, said method comprising:
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preparing a first mixture of labeled nucleotide chain fragments, each labeled chain extending from a common end adjacent to said primary nucleotide and extending to a nucleotide at a site ranging from said primary through said nth nucleotide, said first mixture containing labeled fragments of all lengths from that terminating at said primary nucleotide to that terminating at said nth nucleotide; preparing a second mixture of labeled nucleotide chain fragments, the labeled chain fragments extending from said common end and terminating at each position from said primary nucleotide through said nth nucleotide wherever a first of the four nucleotides occurs and wherever a second of the four nucleotides occurs; preparing a third mixture of labeled nucleotide chain fragments, the labeled chain fragments extending from said common end and terminating at each position from said primary nucleotide through said nth nucleotide wherever said first nucleotide occurs and wherever a third of the four nucleotides occurs; electrophoresing said mixtures of fragments on a gel that separates said fragments according to their length, said fragments being applied to said gel to run in side-by-side lanes; visualizing bands of labeled nucleotide fragments on said lanes of said gel; and reading the nucleotide sequence according to the band patterns in the direction normal to the lanes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification