US5093245A
Expired / Lapsed
US
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Labeling by simultaneous ligation and restriction
Assignee: Applied Biosystems LLC
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Filing: 1988-01-26
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Granted: 1992-03-03
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Expires: 2008-01-26
Abstract
Abstract Termini of restricted double-stranded DNA fragments are modified by ligating the fragments with terminal phosphate-free double-stranded oligonucleotides having a complementary terminus in the presence of a restriction enzyme and a ligase, where joining of the complementary ends results in loss of the restriction enzyme recognition sequence.
Inventors
- 👤 Douglas H. Keith
- 👤 Mel N. Kronick
- 👤 Lincoln J. McBride
- 👤 Norman M. Whiteley
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 19:59
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: 15
Claim 1
Independent Claim
109 words
A method for extending DNA fragments with a ligand comprising: combining in a reaction mixture said fragments in the simultaneous presence of ligase and a restriction enzyme which cuts in or near the recognition site, with dsDNA comprising said ligand and having a terminus complementary to a terminus of said fragments, wherein said fragments have predetermined termini as a result of digestions with said restriction enzyme, said fragments have terminal phosphates and said complementary termini of said dsDNA lack a terminal phosphate and ligation of said dsDNA results in sequence different from the sequence recognized and cleaved by said restriction enzyme, whereby said fragments and dsDNA are covalently joined.
Claim 2
Independent Claim
109 words
A method for extending DNA fragments with a ligand comprising: combining in a reaction mixture said fragments in the simultaneous presence of ligase and a restriction enzyme which cuts in or near the recognition site, with dsDNA comprising said ligand and having a terminus complementary to a terminus of said fragments, wherein said fragments have predetermined termini as a result of digestions with said restriction enzyme, said fragments have terminal phosphates and said complementary termini of said dsDNA lack a terminal phosphate and ligation of said dsDNA results in sequence different from the sequence recognized and cleaved by said restriction enzyme, whereby said fragments and dsDNA are covalently joined.
Claim 3
Dependent Claim
18 words
A method according to claim 1, wherein said ligand is a label capable of providing a detectable signal.
Claim 4
Dependent Claim
15 words
A method according to claim 1, wherein said ligand is a DNA sequence of interest.
Claim 5
Dependent Claim
52 words
A method according to claim 3, including the additional steps of: selecting a fraction of said covalently joined fragments and dsDNA: adding to said selected fraction a DNA polymerase, nucleotide triphosphates and sequences complementary to the strands of said dsDNA: and carrying out a polymerase chain reaction to amplify said selected fraction.
Claim 6
Dependent Claim
14 words
A method according to claim 4, wherein said selecting is size selection by electrophoresis.
Claim 7
Dependent Claim
33 words
A method according to claim 1, wherein said DNA fragments are obtained by adding precursor DNA to be fragmented to said reaction mixture, whereby said precursor DNA is cleaved by said restriction enzyme.
Claim 8
Independent Claim
119 words
A method for labeling DNA fragments with a ligand or radionuclide comprising: combining said fragments in the simultaneous presence of ligase and a restriction enzyme which cuts in or near the recognition site, with ligand or radionuclide labeled dsDNA of fewer than about 200 bp and having a terminus complementary to a terminus of said fragments, wherein said fragments have predetermined termini as a result of digestion with said restriction enzyme, said fragments have terminal phosphates and said complementary termini of said dsDNA lack a terminal phosphate and ligation of said dsDNA results in a sequence different from the sequence recognized and cleaved by said restriction enzyme, whereby said fragments and dsDNA are covalently joined to label said fragments.
Claim 9
Independent Claim
119 words
A method for labeling DNA fragments with a ligand or radionuclide comprising: combining said fragments in the simultaneous presence of ligase and a restriction enzyme which cuts in or near the recognition site, with ligand or radionuclide labeled dsDNA of fewer than about 200 bp and having a terminus complementary to a terminus of said fragments, wherein said fragments have predetermined termini as a result of digestion with said restriction enzyme, said fragments have terminal phosphates and said complementary termini of said dsDNA lack a terminal phosphate and ligation of said dsDNA results in a sequence different from the sequence recognized and cleaved by said restriction enzyme, whereby said fragments and dsDNA are covalently joined to label said fragments.
Claim 10
Dependent Claim
11 words
A method according to claim 7, wherein said ligand is biotin.
Claim 11
Dependent Claim
16 words
A method according to claim 7, wherein said ligand is a radionuclide, fluorescer or an enzyme.
Claim 12
Dependent Claim
12 words
A method according to claim 7, wherein said termini have cohesive ends.
Claim 13
Dependent Claim
33 words
A method according to claim 7, wherein said fragments are obtained by the step of: digesting genomic DNA with at least one restriction enzyme to provide fragments of less than about 200 kbp.
Claim 14
Dependent Claim
112 words
A labeling composition for binding to a restricted dsDNA fragment resulting in loss of the restriction recognition site by simultaneous restriction and ligation, said composition comprising: a bridging strand and first and second strands hybridized to said bridging strand, wherein said strands hybridize contiguously to said bridging strand; said first strand is a labeling strand providing for a double-stranded DNA sequence of interest and a fluorescent label; said second strand is an adapter strand hybridizing to said bridging strand to provide a cohesive end or blunt end with said bridging strand, and wherein said adapter strand contains a particular sequence necessary to destroy the recognition site of a restriction enzyme of interest.
Claim 15
Dependent Claim
112 words
A labeling composition for binding to a restricted dsDNA fragment resulting in loss of the restriction recognition site by simultaneous restriction and ligation, said composition comprising: a bridging strand and first and second strands hybridized to said bridging strand, wherein said strands hybridize contiguously to said bridging strand; said first strand is a labeling strand providing for a double-stranded DNA sequence of interest and a fluorescent label; said second strand is an adapter strand hybridizing to said bridging strand to provide a cohesive end or blunt end with said bridging strand, and wherein said adapter strand contains a particular sequence necessary to destroy the recognition site of a restriction enzyme of interest.
References Cited (Backward Citations)
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