US5124247A
Expired / Lapsed
US
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Process for sequencing nucleic acids
Assignee: European Molecular Biology Laboratory
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Filing: 1989-12-11
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Granted: 1992-06-23
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Expires: 2009-12-11
Abstract
Abstract For the sequencing of nucleic acids by enzymatic extension of an oligonucleotide primer in the presence of a polymerase, the four deoxyribonucleoside triphosphates, one of the four dideoxynucleoside triphosphates and the nucleic acid to be sequenced as template in each of four different preparations, labelling of the nucleic acid fragments which form and which are dependent on the dideoxynucleoside triphosphate used, separation by gel electrophoresis of the preparations containing the fragments and detection of the sequence via the label, at least two of the dideoxynucleoside triphosphates are used in different amounts together in one preparation and the fragments are differentiated by the intensity of the labelling signal of the bands in the gel or in another separation procedure.
Inventors
- 👤 Wilhelm Ansorge
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 05:43
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C07; C07H; C07H21/00; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
113 words
Method for sequencing a nucleic acid comprising: (a) combining (i) an oligonucleotide primer, (ii) a nucleic acid to be sequenced, (iii) four deoxyribonucleoside triphosphates, (iv) a polymerase, and (v) at least three dideoxyribonucleoside triphosphates in different amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced; (b) labelling the nucleic acid fragments formed; (c) separating the nucleic acid fragments by gel electrophoresis; and (d) determining nucleic acid sequence by determination of position of incorporated dideoxynucleoside triphosphates in said labelled nucleic acid fragments, wherein said dideoxynucleoside triphosphates are differentiated from each other by intensity of the label in the nucleic acid sequence.
Claim 2
Dependent Claim
113 words
Method for sequencing a nucleic acid comprising: (a) combining (i) an oligonucleotide primer, (ii) a nucleic acid to be sequenced, (iii) four deoxyribonucleoside triphosphates, (iv) a polymerase, and (v) at least three dideoxyribonucleoside triphosphates in different amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced; (b) labelling the nucleic acid fragments formed; (c) separating the nucleic acid fragments by gel electrophoresis; and (d) determining nucleic acid sequence by determination of position of incorporated dideoxynucleoside triphosphates in said labelled nucleic acid fragments, wherein said dideoxynucleoside triphosphates are differentiated from each other by intensity of the label in the nucleic acid sequence.
Claim 3
Dependent Claim
39 words
Method of claim 1, wherein said three dideoxynucleoside triphosphates are combined with (i), (ii), (iii) and (iv) in two preparations, a first preparation containing different amounts of two dideoxynucleotide triphosphates and a second preparation containing a third dideoxynucleotide triphosphate.
Claim 4
Dependent Claim
19 words
Method of claim 1, comprising combining four different dideoxynucleoside triphosphates in different amounts with (i), (ii), (iii) and (iv).
Claim 5
Dependent Claim
23 words
Method of claim 3, wherein different amounts of said four dideoxynucleotide triphosphates are combined with (i), (ii), (iii) and (iv) in one preparation.
Claim 6
Dependent Claim
46 words
Method of claim 3, wherein different amounts of two different dideoxynucleotides are combined with (i), (ii), (iii) and (iv) in a first preparation and different amounts of a third and a fourth dideoxynucleotide triphosphate are combined with (i), (ii), (iii) and (iv) in a second preparation.
Claim 7
Dependent Claim
23 words
Method of claim 2, wherein the two said dideoxyribonucleoside triphosphates present in said first preparation are present in a ratio of about 2:1.
Claim 8
Dependent Claim
18 words
Method of claim 3, wherein said four dideoxynucleotide triphosphates are used in a ratio of at least 4:3:2:1.
Claim 9
Dependent Claim
15 words
Method of claim 1, wherein said nucleic acid fragments are labelled with a fluorescent dye.
Claim 10
Dependent Claim
19 words
Method of claim 8, comprising labelling said nucleic acid fragments by coupling said fluorescent dye to said oligonucleotide primer.
Claim 11
Dependent Claim
21 words
Method of claim 9, wherein said fluorescent dye is bound, via a linker, to a 5'-phosphate group of said oligonucleotide primer.
Claim 12
Dependent Claim
25 words
Method of claim 8, wherein said fluorescent dye is bound, via a linker to at least one deoxynucleoside triphosphate or at least one dideoxynucleoside triphosphate.
Claim 13
Dependent Claim
17 words
Method of claim 11, wherein said fluorescent dye is bound via a linker to one deoxynucleoside triphosphate.
Claim 14
Dependent Claim
20 words
Method of claim 11, wherein said fluroescent dye is bound, via a linker, to a C5 position of a pyrimidine.
Claim 15
Dependent Claim
23 words
Method of claim 11, wherein said fluorescent dye is bound, via a linker, to a N7, C7 or C8 position of a purine.
Claim 16
Dependent Claim
38 words
Method of claim 10 or 11, wherein said linker is a straight chain or branched amino hydrocarbon or a straight chain or branched mercapto hydrocarbon, wherein said hydrocarbon contains an unbranched chain having 3 or more carbon atoms.
Claim 17
Dependent Claim
14 words
Method of claim 15, wherein said linker is an aminoalkyl, aminoalkenyl or aminoalkynyl group.
Claim 18
Dependent Claim
11 words
Method of claim 1, comprising sequencing a single stranded nucleic acid.
Claim 19
Dependent Claim
11 words
Method of claim 1, comprising sequencing a double stranded nucleic acid.
Claim 20
Dependent Claim
16 words
Method of claim 8, wherein said fluorescent dye is fluorscein, a fluorescent fluorescin analogue, or rhodamine.
Claim 21
Dependent Claim
28 words
Method of claim 2 or 5, wherein said two dideoxynucleoside triphosphates are used in a ratio of X:1, wherein X is a whole number from 5 to 7.
Claim 22
Dependent Claim
19 words
Method of claim 3 or 4, wherein said four different dideoxynucleotide triphosphates are used in a ratio of 16:8:4:1.
Claim 23
Dependent Claim
26 words
Method of claim 1, wherein said polymerase is Klenow fragment of DNA polymerase I, T7 DNA polymerase, modified T7 DNA polymerase, Taq polymerase or reverse transcriptase.
Claim 24
Dependent Claim
11 words
Method of claim 1, wherein said polymerase is T7 DNA polymerase.
Claim 25
Dependent Claim
52 words
Method of claim 1, further comprising subjecting said nucleic acid sequence to be determined to polymerase chain reaction prior to determining its sequence to increase the amount of said nucleic acid sequence, separating double stranded nucleic acids thus produced, and using one of the double strands to determine said nucleic acid sequence.
Claim 26
Dependent Claim
16 words
Method of claim 8, further comprising exciting said fluorescent dye with a laser in step (d).
References Cited (Backward Citations)
US4683195A
Cited
US4683195B1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-06-23 | 1995-12-23 | 1996-06-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-06-23 | 1999-12-23 | 2000-06-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-06-23 | 2003-12-23 | 2004-06-23 | $7700.00 | Window Closed / Lapsed |