US4865968A
Expired / Lapsed
US
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DNA sequencing
Assignee: Salk Institute for Biological Studies
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Filing: 1985-04-01
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Granted: 1989-09-12
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Expires: 2005-04-01
Abstract
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.
Inventors
- 👤 Leslie E. Orgel
- 👤 James W. Patrick
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 02:01
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; Y; Y10; Y10S; Y10S435/00; Y10S435/803
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
242 words
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.
Claim 2
Independent Claim
242 words
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.
Claim 3
Dependent Claim
88 words
A method according to claim 1 wherein said nucleotide chain fragments are prepared in a plurality of reactions by obtaining a single-stranded nucleotide chain to be used as a template, annealing said single-stranded template nucleotide to a single-stranded primer, and producing a complementary copy of said template with a mixture that includes a polymerase, the four nucleoside triphosphates, and selected analogs which each correspond to one of the four nucleoside triphosphates and acts as a substrate for said polymerase but terminates chain elongation at the position of incorporation.
Claim 4
Dependent Claim
19 words
A method according to claim 2 wherein said mixtures each contains at least one nucleoside triphosphate labeled for visualization.
Claim 5
Dependent Claim
79 words
A method according to claim 1 wherein said nucleotide chain fragments are prepared by obtaining a double-stranded nucleotide chain, labeling said double-stranded nucleotide chain at a single 5' end, and nick-translating said labeled nucleotide chain in a plurality of reaction mixtures, each mixture including nick-translation enzymes, the four nucleoside triphosphates and selected analogs which each corresponds to one of the four nucleotides and acts as a substrate for said polymerase but terminates chain elongation at the position of incorporation.
Claim 6
Dependent Claim
56 words
A method according to claim 1 wherein said mixture of chain fragments is obtained by labeling a single-stranded nucleotide chain at one end, and in a plurality of reaction mixtures, cleaving aliquots of said labeled chain under conditions which cleave the same on the average of once each chain at specific ones of the four nucleotides.
Claim 7
Dependent Claim
52 words
A method according to claim 1 wherein said first mixture is applied to run in a first lane, said first lane flanked on one side by a second land to which said second mixture is applied and on the other side by a third lane to which said third mixture is applied.
Claim 8
Dependent Claim
43 words
A method according to claim 1 wherein four samples are prepared, each containing labeled nucleotide chain fragments terminating at the site of a different one of the four nucleotides and preparing said first, second and third mixtures from aliquots of said four samples.
Claim 9
Dependent Claim
43 words
A method according to claim 4 wherein four samples are prepared, each containing labeled nucleotide chain fragments terminating at the site of a different one of the four nucleotides and preparing said first, second and third mixtures from aliquots of said four samples.
Claim 10
Dependent Claim
43 words
A method according to claim 5 wherein four samples are prepared, each containing labeled nucleotide chain fragments terminating at the site of the different one of the four nucleotides and preparing said first, second and third mixtures from aliquots of said four samples.
Claim 11
Dependent Claim
52 words
A method according to claim 2 wherein said first mixture is applied to run in a first lane, said first lane flanked on one side by a second lane to which said second mixture is applied and on the other side by a third lane to which said third mixture is applied.
Claim 12
Dependent Claim
52 words
A method according to claim 3 wherein said first mixture is applied to run in a first lane, said first lane flanked on one side by a second lane to which said second mixture is applied and on the other side by a third lane to which said third mixture is applied.
Claim 13
Dependent Claim
52 words
A method according to claim 4 wherein said first mixture is applied to run in a first lane, said first lane flanked on one side by a second lane to which said second mixture is applied and on the other side by a third lane to which said third mixture is applied.
Claim 14
Dependent Claim
52 words
A method according to claim 5 wherein said first mixture is applied to run in a first lane, said first lane flanked on one side by a second lane to which said second mixture is applied and on the other side by a third lane to which said third mixture is applied.
Claim 15
Dependent Claim
52 words
A method according to claim 7 wherein said first mixture is applied to run in a first lane, said first lane flanked on one side by a second lane to which said second mixture is applied and on the other side by a third lane to which said third mixture is applied.
Claim 16
Dependent Claim
43 words
A method according to claim 2 wherein four samples are prepared, each containing labeled nucleotide chain fragments terminating at the site of a different one of the four nucleotides and preparing said first, second and third mixtures from aliquots of said four samples.
Claim 17
Dependent Claim
43 words
A method according to claim 3 wherein four samples are prepared, each containing labeled nucleotide chain fragments terminating at the site of a different one of the four nucleotides and preparing said first, second and third mixtures from aliquots of said four samples.
References Cited (Backward Citations)
EP0115777A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-09-11 | 1993-03-13 | 1993-09-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-09-11 | 1997-03-13 | 1997-09-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-09-11 | 2001-03-13 | 2001-09-12 | $7700.00 | Window Closed / Lapsed |