US6007987A
Expired / Lapsed
US
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Positional sequencing by hybridization
Assignee: Boston University
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Filing: 1996-10-15
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Granted: 1999-12-28
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Expires: 2016-10-15
Abstract
Abstract This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.
Inventors
- 👤 Charles R. Cantor
- 👤 Marek Przetakiewicz
- 👤 Takeshi Sano
- 👤 Cassandra L. Smith
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 23:16
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6834; C12Q1/6837; B; B01; B01J; B01J2219/00; B01J2219/00274; B01J2219/00583; B01J2219/00603; B01J2219/00605; B01J2219/0061; B01J2219/00612; B01J2219/00623; B01J2219/00626; B01J2219/0063; B01J2219/00632; B01J2219/00637; B01J2219/00659; B01J2219/00718; B01J2219/0072; B01J2219/00722; C40; C40B; C40B40/00; C40B40/04; C40B40/06
Claims Analysis
Total Claims: 9
Claim 1
Independent Claim
172 words
A method of forming an array of probes comprising the steps of: i) providing: a) a restriction enzyme selected from the group consisting of restriction enzymes which produce 5'-overhangs and restriction enzymes which produce 3'-overhangs, b) an array of single-stranded nucleic acids each containing a constant sequence at the 3'-terminus, another constant sequence at the 5'-terminus, and a random internal sequence between about 3-5 nucleotides in length flanked by the cleavage sites of said restriction enzyme, c) a plurality of primers each complimentary to a portion of the constant sequence of the 3'-terminus of said single-stranded nucleic acids of said array, and d) a polymerase; ii) hybridizing said primers to said single-stranded nucleic acids of said array to create hybridized primers; iii) extending the sequence of each hybridized primer by polymerization with said polymerase to create extended hybrids; and iv) cleaving said extended hybrids with said restriction enzyme to form an array of probes with a double-stranded portion at one terminus, a single-stranded portion containing the random sequences at the opposite terminus.
Claim 2
Independent Claim
172 words
A method of forming an array of probes comprising the steps of: i) providing: a) a restriction enzyme selected from the group consisting of restriction enzymes which produce 5'-overhangs and restriction enzymes which produce 3'-overhangs, b) an array of single-stranded nucleic acids each containing a constant sequence at the 3'-terminus, another constant sequence at the 5'-terminus, and a random internal sequence between about 3-5 nucleotides in length flanked by the cleavage sites of said restriction enzyme, c) a plurality of primers each complimentary to a portion of the constant sequence of the 3'-terminus of said single-stranded nucleic acids of said array, and d) a polymerase; ii) hybridizing said primers to said single-stranded nucleic acids of said array to create hybridized primers; iii) extending the sequence of each hybridized primer by polymerization with said polymerase to create extended hybrids; and iv) cleaving said extended hybrids with said restriction enzyme to form an array of probes with a double-stranded portion at one terminus, a single-stranded portion containing the random sequences at the opposite terminus.
Claim 3
Dependent Claim
17 words
The method of claim 1 wherein the nucleic acids are each between about 10-50 nucleotides in length.
Claim 4
Dependent Claim
36 words
The method of claim 1 wherein the array of probes is fixed to a solid support and the solid support which is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and chips.
Claim 5
Dependent Claim
11 words
An array of probes created by the method of claim 1.
Claim 6
Independent Claim
175 words
A method of forming an array of probes comprising the steps of: i) providing: a) an array of single-stranded nucleic acids each containing a constant sequence at the 3'-terminus, another constant sequence at the 5'-terminus, and random internal sequence at the 3'-terminus; b) a plurality of primers with a sequence complementary to the constant sequence at the 3'-terminus of said single-stranded nucleic acids, and c) a restriction enzyme selected from the group consisting of restriction enzymes which produce 5'-overhangs and restriction enzymes which produce 3'-overhangs; ii) hybridizing said primers to said array to form hybrids; iii) enzymatically extending the primers of said hybrids to form full-length hybrids; iv) cloning said full-length hybrids into vectors to create cloned sequences; v) amplifying said cloned sequences by multiple polymerase chain reactions to generate amplified sequences; and vi) cleaving said amplified sequences with said restriction enzyme to form an array of probes with a double-stranded portion at one terminus and a single-stranded portion containing the random sequence at the opposite terminus, wherein the array of probes have 5'-or 3'-overhangs.
Claim 7
Independent Claim
175 words
A method of forming an array of probes comprising the steps of: i) providing: a) an array of single-stranded nucleic acids each containing a constant sequence at the 3'-terminus, another constant sequence at the 5'-terminus, and random internal sequence at the 3'-terminus; b) a plurality of primers with a sequence complementary to the constant sequence at the 3'-terminus of said single-stranded nucleic acids, and c) a restriction enzyme selected from the group consisting of restriction enzymes which produce 5'-overhangs and restriction enzymes which produce 3'-overhangs; ii) hybridizing said primers to said array to form hybrids; iii) enzymatically extending the primers of said hybrids to form full-length hybrids; iv) cloning said full-length hybrids into vectors to create cloned sequences; v) amplifying said cloned sequences by multiple polymerase chain reactions to generate amplified sequences; and vi) cleaving said amplified sequences with said restriction enzyme to form an array of probes with a double-stranded portion at one terminus and a single-stranded portion containing the random sequence at the opposite terminus, wherein the array of probes have 5'-or 3'-overhangs.
Claim 8
Dependent Claim
37 words
The method of claim 5 wherein the array of probes is fixed to a solid support and the solid support is selected from the group consisting of plastics, ceramics, metals, resins, polymers, films, gels, membranes and chips.
Claim 9
Dependent Claim
11 words
An array of probes created by the method of claim 5.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2002-12-27 | 2003-06-28 | 2003-12-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-12-27 | 2007-06-28 | 2007-12-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-12-27 | 2011-06-28 | 2011-12-28 | $7700.00 | Window Closed / Lapsed |