US5366877A
Expired / Lapsed
US
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Restriction/ligation labeling for primer initiated multiple copying of DNA ssequences
Assignee: Applied Biosystems LLC
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Filing: 1990-10-22
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Granted: 1994-11-22
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Expires: 2010-10-22
Abstract
Abstract Sample DNA is analyzed by joining dsDNA sample fragments to labeling moieties having a primer binding sequence, to provide labeled dsDNA. After denaturation of the labeled dsDNA, strands binding to a probe are separated, conveniently using particles and a specific binding pair, followed by amplification of the sample strands and analysis and/or isolation of the amplified strands.
Inventors
- 👤 Douglas H. Keith
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 20:28
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6844; C12Q1/6853; C12Q1/6855; C12Q1/6858; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
198 words
A method for amplifying dsDNA comprising a target sequence., said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a 5'-3' first strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.
Claim 2
Independent Claim
198 words
A method for amplifying dsDNA comprising a target sequence., said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a 5'-3' first strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.
Claim 3
Dependent Claim
54 words
A method according to claim 1, wherein said covalently joining comprises: ligating said first strands to the 5'-termini of said dsDNA fragment strands to produce a first strand ligated product; removing said ligaid strand by displacement; extending the 3'-termini of said dsDNA strands to produce said ligaid sequences complementary to said first strand sequences.
Claim 4
Dependent Claim
78 words
A method according to claim 1, wherein said covalently joining comprises: ligating said first strands to the 5'-termini of said dsDNA fragment strands to produce a first strand ligated product; displacing said ligaid or truncated ligaid strand by denaturation and hybridization with a second ligaid strand, wherein said second ligaid strand is phosphorylated at the 5'-terminus not later than before ligating; and ligating said ligaid to the 3'-terminus of said dsDNA strands in registry with said ligaid strand.
Claim 5
Dependent Claim
32 words
A method according to claim 1, wherein said labeling moieties comprise a ligaid phosphorylated at the 5'-terminus, and said phosphorylated ligaid is ligated to the dsDNA strand in registry with said ligaid.
Claim 6
Dependent Claim
29 words
A method according to claim 1, comprising the additional step of: separating said amplified dsDNA by size to identify the size of the dsDNA fragment comprising said target sequence.
Claim 7
Dependent Claim
27 words
A method according to claim 1, wherein said separation is by means of paramagnetic particles, wherein said labeled single strands are non-covalently bound to said paramagnetic particles.
Claim 8
Dependent Claim
29 words
A method according to claim 6, wherein said separation is by means of a probe complementary to said target sequence and comprising biotin and said paramagnetic particles comprise strept/avidin.
Claim 9
Independent Claim
267 words
A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a member of a specific binding pair to produce probe bound strands, combining said probe bound strands with the complementary member of said specific binding pair bound to a solid support, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.
Claim 10
Independent Claim
267 words
A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a member of a specific binding pair to produce probe bound strands, combining said probe bound strands with the complementary member of said specific binding pair bound to a solid support, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.
Claim 11
Dependent Claim
13 words
A method according to claim 8, wherein said solid support is paramagnetic particles.
Claim 12
Dependent Claim
28 words
A method according to claim 8, wherein said ligaid or truncated ligaid is displaced with a ligaid comprising said primer binding site sequence and phosphorylated at the 5'-terminus.
Claim 13
Independent Claim
246 words
A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced and substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer sequence proximal to the 3'-terminus of said labeled single stranded DNA and its complementary sequence 5' to said single stranded DNA; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a solid support to produce probe bound strands, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.
Claim 14
Independent Claim
246 words
A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced and substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer sequence proximal to the 3'-terminus of said labeled single stranded DNA and its complementary sequence 5' to said single stranded DNA; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a solid support to produce probe bound strands, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.
Claim 15
Dependent Claim
13 words
A method according to claim 11, wherein said solid support is paramagnetic particles.
Claim 16
Dependent Claim
26 words
A method according to claim 11, wherein said ligaid or truncated ligaid is displaced with a ligaid comprising said primer sequence and phosphorylated at the 5'-terminus.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-11-21 | 1998-05-23 | 1998-11-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-11-21 | 2002-05-23 | 2002-11-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-11-21 | 2006-05-23 | 2006-11-22 | $7700.00 | Window Closed / Lapsed |