US5405746A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method of sequencing DNA
Assignee: Qiagen GmbH
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Filing: 1993-09-22
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Granted: 1995-04-11
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Expires: 2013-09-22
Abstract
Abstract The invention relates to a method of sequencing target DNA in which said DNA is provided in double stranded form immobilized on a solid support via one terminus of one of the two strands thereof and is then subjected to strand separation whereby the unattached strand is removed prior to sequencing the immobilized strand. The target DNA, e.g genomic DNA, may initially be amplified by the PCR method using at least one oligonucleotide primer which is either attached to a solid support or carries means for attachment to a solid support.
Inventors
- 👤 Mathias Uhlen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 06:45
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12Q1/6874; C12Q1/6806
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
73 words
A method for solid-phase sequencing of one strand of double-stranded DNA comprising the steps of: (A) providing said double-stranded DNA; (B) immobilizing on a solid support one terminus of one of the two strands of said double-stranded DNA; (C) subjecting said immobilized double-stranded DNA to strand separation, thereby providing an immobilized strand and an unattached strand; (D) removing said unattached strand, thereby providing an immobilized single-stranded DNA; (E) sequencing said immobilized single-stranded DNA.
Claim 2
Independent Claim
73 words
A method for solid-phase sequencing of one strand of double-stranded DNA comprising the steps of: (A) providing said double-stranded DNA; (B) immobilizing on a solid support one terminus of one of the two strands of said double-stranded DNA; (C) subjecting said immobilized double-stranded DNA to strand separation, thereby providing an immobilized strand and an unattached strand; (D) removing said unattached strand, thereby providing an immobilized single-stranded DNA; (E) sequencing said immobilized single-stranded DNA.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein strand separation is effected by melting.
Claim 4
Dependent Claim
35 words
The method of claim 1 wherein prior to step (B), a functional group which binds to the solid support is introduced at only one terminus of one of the strands of the double stranded DNA.
Claim 5
Dependent Claim
80 words
The method of claim 1 wherein the double stranded DNA is a plasmid containing a target sequence; the plasmid is cleaved at a restriction site outside the target sequence; a functional group is introduced at at least one terminus of one strand only of said DNA; the functionality DNA is cleaved at a further restriction site outside the target sequence; and the functionalized target sequence is contacted with a solid support carrying a substance with affinity for said functional group.
Claim 6
Dependent Claim
84 words
The method of claim 1 wherein the double stranded DNA is a plasmid containing a target sequence; the plasmid DNA is cleaved at a restriction site outside the target sequence and at one or more restriction sites within the target sequence; and oligonucleotides are ligated at both termini of the plasmid DNA generated by said cleavage, wherein one of said oligonucleotides carries a functional group which binds to a solid support and the other oligonucleotide is capable of hybridizing to a general sequencing primer,
Claim 7
Dependent Claim
33 words
The method of claim 1 wherein the immobilized double stranded DNA is produced by the polymerase chain reaction method using at least one oligonucleotide primer carrying means for attachment to a solid support.
Claim 8
Dependent Claim
24 words
The method of claim 6 wherein the immobilized double stranded DNA is produced by amplification of genomic DNA by the polymerase chain reaction method.
Claim 9
Dependent Claim
18 words
The method of claim 1 wherein said immobilized single stranded DNA is sequenced by the primer extension method,
Claim 10
Dependent Claim
25 words
The method of claim 3 wherein said functional group is a biotin, avidin, streptavidin or thiol group and said solid support carries groups binding thereto.
Claim 11
Dependent Claim
18 words
The method of claim 1 wherein the solid support is in particulate form or is a microtitre well.
Claim 12
Dependent Claim
15 words
The method of claim 1 wherein stand separation is effectuated by treatment with aqueous alkali.
Claim 13
Dependent Claim
11 words
The method of claim 9, wherein said functional group is biotin.
Claim 14
Dependent Claim
16 words
The method of claim 11 wherein said strand separation is effectuated by treatment with 0.15M NaOH.
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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) | 1998-04-10 | 1998-10-10 | 1999-04-11 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-04-10 | 2002-10-10 | 2003-04-11 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-04-10 | 2006-10-10 | 2007-04-11 | $7700.00 | Window Closed / Lapsed |