US5503980A
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US
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Positional sequencing by hybridization
Assignee: Boston University
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Filing: 1994-10-17
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Granted: 1996-04-02
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Expires: 2014-10-17
Abstract
Abstract This invention is directed to methods for determining a nucleotide sequence of a nucleic acid using positional sequencing by hybridization, and to the creation of nucleic acids probes which may be used with these methods. This invention is also directed to diagnostic aids for analyzing the nucleic acid composition and content of biological samples, including samples derived from medical and agricultural sources.
Inventors
- 👤 Charles R. Cantor
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 21:40
CPC Classifications
B; B01; B01J; B01J19/00; B01J19/0046; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6834; C12Q1/6837; C40; C40B; C40B80/00; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00497; B01J2219/00527; B01J2219/00529; B01J2219/00583; B01J2219/0059; B01J2219/00603; B01J2219/00605; B01J2219/0061; B01J2219/00612; B01J2219/00623; B01J2219/00626; B01J2219/00632; B01J2219/00637; B01J2219/00646; B01J2219/00648; B01J2219/00659; B01J2219/00718; B01J2219/0072; B01J2219/00722; C40B40/00; C40B40/04; C40B40/06
Claims Analysis
Total Claims: 44
Claim 1
Independent Claim
96 words
A method for determining a nucleotide sequence of a nucleic acid target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion, a single stranded portion, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target which is at least partly single stranded to one or more of the nucleic acid probes; and (c) determining the nucleotide sequence of the target which is hybridized to the single stranded portion of any probe.
Claim 2
Independent Claim
96 words
A method for determining a nucleotide sequence of a nucleic acid target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion, a single stranded portion, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target which is at least partly single stranded to one or more of the nucleic acid probes; and (c) determining the nucleotide sequence of the target which is hybridized to the single stranded portion of any probe.
Claim 3
Independent Claim
104 words
A method for determining a nucleotide sequence of a nucleic acid target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion, a single stranded portion, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target which is at least partly single stranded to one or more of the nucleic acid probes; (c) ligating the hybridized target to the probe; and (d) determining the nucleotide sequence of the target which is hybridized to the single stranded portion of any probe.
Claim 4
Independent Claim
104 words
A method for determining a nucleotide sequence of a nucleic acid target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion, a single stranded portion, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target which is at least partly single stranded to one or more of the nucleic acid probes; (c) ligating the hybridized target to the probe; and (d) determining the nucleotide sequence of the target which is hybridized to the single stranded portion of any probe.
Claim 5
Dependent Claim
21 words
The method of claim 1 wherein the nucleic acid target is selected from the group consisting of DNA, RNA and PNA.
Claim 6
Dependent Claim
21 words
The method of claim 1 wherein the nucleic acid probe is selected from the group consisting of DNA, RNA and PNA.
Claim 7
Dependent Claim
24 words
The method of claim 1 wherein the variable nucleotide sequence within the single stranded portion of the probe is about 4-20 nucleotides in length.
Claim 8
Dependent Claim
22 words
The method of claim 1 wherein the double stranded portion of each probe is constant and is about 10-30 nucleotides in length.
Claim 9
Dependent Claim
14 words
The method of claim 1 wherein the target is about 30-300 nucleotides in length.
Claim 10
Dependent Claim
18 words
The method of claim 1 wherein the set of nucleic acid probes is fixed to a solid support.
Claim 11
Dependent Claim
23 words
The method of claim 8 wherein the solid support is a plastic, a ceramic, a metal, a resin, a gel, or a membrane.
Claim 12
Dependent Claim
20 words
The method of claim 8 wherein the solid support is a two-dimensional or three-dimensional matrix with multiple probe binding sites.
Claim 13
Dependent Claim
15 words
The method of claim 10 wherein the two-dimensional or three-dimensional matrix is a hybridization chip.
Claim 14
Dependent Claim
14 words
The method of claim 1 wherein the target is labeled with a detectable label.
Claim 15
Dependent Claim
35 words
The method of claim 12 wherein the detectable label is a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, an electric charge, or a spatial chemical.
Claim 16
Dependent Claim
29 words
The method of claim 12 wherein the target is labeled with a first detectable label at a terminal site and a second different detectable label at an internal site.
Claim 17
Dependent Claim
17 words
The method of claim 14 wherein the first and second detectable labels are chromatic or fluorescent chemicals.
Claim 18
Dependent Claim
16 words
The method of claim 14 wherein the first and second labels are detectable by mass spectrometry.
Claim 19
Independent Claim
109 words
A method for determining a nucleotide sequence of a target nucleic acid by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion at a 3'-terminus, a single stranded portion at a 5'-terminus, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target nucleic acid which is at least partly single stranded to the set; (c) enzymatically extending a strand of the probe using the hybridized target as a template; and (d) determining the nucleotide sequence of the single stranded portion of the target nucleic acid.
Claim 20
Independent Claim
109 words
A method for determining a nucleotide sequence of a target nucleic acid by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion at a 3'-terminus, a single stranded portion at a 5'-terminus, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target nucleic acid which is at least partly single stranded to the set; (c) enzymatically extending a strand of the probe using the hybridized target as a template; and (d) determining the nucleotide sequence of the single stranded portion of the target nucleic acid.
Claim 21
Independent Claim
117 words
A method for determining a nucleotide sequence of a target nucleic acid by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion at a 3'-terminus, a single stranded portion at a 5'-terminus, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target nucleic acid which is at least partly single stranded to the set; (c) ligating the hybridized target to the probe; (d) enzymatically extending a strand of the probe using the hybridized target as a template; and (e) determining the nucleotide sequence of the single stranded portion of the target nucleic acid.
Claim 22
Independent Claim
117 words
A method for determining a nucleotide sequence of a target nucleic acid by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion at a 3'-terminus, a single stranded portion at a 5'-terminus, and a variable sequence within the single stranded portion which is determinable; (b) hybridizing the target nucleic acid which is at least partly single stranded to the set; (c) ligating the hybridized target to the probe; (d) enzymatically extending a strand of the probe using the hybridized target as a template; and (e) determining the nucleotide sequence of the single stranded portion of the target nucleic acid.
Claim 23
Dependent Claim
26 words
The method of claim 17 wherein the probe is enzymatically extended by DNA polymerase, using one or more dideoxynucleotide triphosphates and one or more deoxynucleotides triphosphates.
Claim 24
Dependent Claim
18 words
The method of claim 17 wherein the probe is enzymatically extended by DNA polymerase using one dideoxynucleotide triphosphate.
Claim 25
Dependent Claim
12 words
The method of claim 17 wherein the probe has a detectable label.
Claim 26
Dependent Claim
35 words
The method of claim 21 wherein the detectable label is a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, a electric charge, or a spatial structure.
Claim 27
Dependent Claim
29 words
The method of claim 17 wherein the target is labeled with a first detectable label at a terminal site and a second different detectable label at an internal site.
Claim 28
Dependent Claim
17 words
The method of claim 23 wherein the first and second detectable labels are chromatic or fluorescent chemicals.
Claim 29
Dependent Claim
16 words
The method of claim 23 wherein the first and second labels are detectable by mass spectrometry.
Claim 30
Independent Claim
115 words
A method for determining a nucleotide sequence of a target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is about 14-50 nucleotides in length and has a double-stranded portion, a single-stranded portion, and a variable nucleotide sequence within the single-stranded portion which is determinable; (b) cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded; (c) hybridizing the single-stranded region of the fragments with the single-stranded region of the probes; (d) identifying the nucleotide sequences of the hybridized portions of the fragments; and (e) comparing the identified nucleotide sequences to determine the nucleotide sequence of the target.
Claim 31
Independent Claim
115 words
A method for determining a nucleotide sequence of a target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is about 14-50 nucleotides in length and has a double-stranded portion, a single-stranded portion, and a variable nucleotide sequence within the single-stranded portion which is determinable; (b) cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded; (c) hybridizing the single-stranded region of the fragments with the single-stranded region of the probes; (d) identifying the nucleotide sequences of the hybridized portions of the fragments; and (e) comparing the identified nucleotide sequences to determine the nucleotide sequence of the target.
Claim 32
Independent Claim
123 words
A method for determining a nucleotide sequence of a target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is about 14-50 nucleotides in length and has a double-stranded portion, a single-stranded portion, and a variable nucleotide sequence within the single-stranded portion which is determinable; (b) cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded; (c) hybridizing the single-stranded region of the fragments with the single-stranded region of the probes; (d) ligating the hybridized fragments to the probes; (e) identifying the nucleotide sequences of the hybridized portions of the fragments; and (f) comparing the identified nucleotide sequences to determine the nucleotide sequence of the target.
Claim 33
Independent Claim
123 words
A method for determining a nucleotide sequence of a target by hybridization comprising the steps of: (a) creating a set of nucleic acid probes wherein each probe is about 14-50 nucleotides in length and has a double-stranded portion, a single-stranded portion, and a variable nucleotide sequence within the single-stranded portion which is determinable; (b) cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded; (c) hybridizing the single-stranded region of the fragments with the single-stranded region of the probes; (d) ligating the hybridized fragments to the probes; (e) identifying the nucleotide sequences of the hybridized portions of the fragments; and (f) comparing the identified nucleotide sequences to determine the nucleotide sequence of the target.
Claim 34
Dependent Claim
14 words
The method of claim 26 wherein cleaving occurs by enzymatic, chemical or physical means.
Claim 35
Dependent Claim
18 words
The method of claim 26 wherein the set of nucleic acid probes is fixed to a solid support.
Claim 36
Dependent Claim
23 words
The method of claim 29 wherein the solid support is a plastic, a ceramic, a metal, a resin, a gel, or a membrane.
Claim 37
Dependent Claim
20 words
The method of claim 29 wherein the solid support is a two-dimensional or three-dimensional matrix with multiple probe binding sites.
Claim 38
Dependent Claim
14 words
The method of claim 26 wherein the target nucleic acid has a detectable label.
Claim 39
Dependent Claim
35 words
The method of claim 32 wherein the detectable label is a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, a electric charge, or a spatial structure.
Claim 40
Dependent Claim
28 words
The method of claim 32 wherein the target nucleic acid has a first detectable label at a terminal site and a second detectable label at an internal site.
Claim 41
Dependent Claim
17 words
The method of claim 34 wherein the first and second detectable labels are chromatic or fluorescent chemicals.
Claim 42
Dependent Claim
16 words
The method of claim 34 wherein the first and second labels are detectable by mass spectrometry.
Claim 43
Independent Claim
98 words
A method for determining a nucleotide sequence within a nucleic acid fragment comprising the steps of: (a) labeling the nucleic acid fragment with a first detectable label at a terminal site and with a second detectable label in a plurality of internal sites; (b) cleaving the labeled nucleic acid fragment into portions; (c) aligning the relative positions of said portions to the nucleic acid fragment by determining the ratio of the first detectable label to the second detectable label; and (d) determining the nucleotide sequence of said portions to determine the nucleotide sequence within said nucleic acid fragment.
Claim 44
Independent Claim
98 words
A method for determining a nucleotide sequence within a nucleic acid fragment comprising the steps of: (a) labeling the nucleic acid fragment with a first detectable label at a terminal site and with a second detectable label in a plurality of internal sites; (b) cleaving the labeled nucleic acid fragment into portions; (c) aligning the relative positions of said portions to the nucleic acid fragment by determining the ratio of the first detectable label to the second detectable label; and (d) determining the nucleotide sequence of said portions to determine the nucleotide sequence within said nucleic acid fragment.
References Cited (Backward Citations)
US5202231A
Cited
US5149625A
Cited
US5073483A
Cited
US5002867A
Cited
US5068176A
Cited
WO1989010977A1
Cited
US5114839A
Cited
EP0392546A2
Cited
US5106727A
Cited
US5112734A
Cited
US5112736A
Cited
US5137806A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1999-04-02 | 1999-10-02 | 2000-04-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-04-02 | 2003-10-02 | 2004-04-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-04-02 | 2007-10-02 | 2008-04-02 | $7700.00 | Window Closed / Lapsed |