US5470705A
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Probe composition containing a binding domain and polymer chain and methods of use
Assignee: Applied Biosystems LLC
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Filing: 1992-04-07
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Granted: 1995-11-28
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Expires: 2012-04-07
Abstract
Abstract Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.
Inventors
- 👤 Paul D. Grossman
- 👤 Steven Fung
- 👤 Steven M. Menchen
- 👤 Sam L. Woo
- 👤 Emily S. Winn-Deen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 15:18
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6825; C12Q1/6827; C12Q1/6844; C12Q1/686; C12Q1/6862; C12Q2600/00; C12Q2600/156; G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
161 words
A method of identifying one or more of a plurality of different sequences in a target polynucleotide, comprising adding to the target polynucleotide, a plurality of sequence-specific probes, each characterized by (a) an oligonucleotide binding polymer having a probe-specific sequence of subunits designed for base-specific binding of the binding polymer to one of the target sequences, under selected binding conditions, and (b) attached to the binding polymer, a polymer chain having a ratio of charge/translational frictional drag which is different from that of the binding polymer, reacting the probes with the target polynucleotide under conditions favoring binding of the probes in a base-specific manner to the target polynucleotide, treating the probes to selectively modify those probes which are bound to the target polynucleotide in a sequence-specific manner, to form modified, labeled probes characterized by (a) a distinctive ratio of charge/translational frictional drag, and (b) a detectable reporter label, and fractionating the modified, labeled probe(s) by capillary electrophoresis in a non-sieving medium.
Claim 2
Independent Claim
161 words
A method of identifying one or more of a plurality of different sequences in a target polynucleotide, comprising adding to the target polynucleotide, a plurality of sequence-specific probes, each characterized by (a) an oligonucleotide binding polymer having a probe-specific sequence of subunits designed for base-specific binding of the binding polymer to one of the target sequences, under selected binding conditions, and (b) attached to the binding polymer, a polymer chain having a ratio of charge/translational frictional drag which is different from that of the binding polymer, reacting the probes with the target polynucleotide under conditions favoring binding of the probes in a base-specific manner to the target polynucleotide, treating the probes to selectively modify those probes which are bound to the target polynucleotide in a sequence-specific manner, to form modified, labeled probes characterized by (a) a distinctive ratio of charge/translational frictional drag, and (b) a detectable reporter label, and fractionating the modified, labeled probe(s) by capillary electrophoresis in a non-sieving medium.
Claim 3
Dependent Claim
37 words
The method of claim 1, wherein the different-sequence binding polymers have substantially the same lengths, as evidenced by the ability of the binding polymers to hybridize to their associated target sequences with the same hybridization reaction kinetics.
Claim 4
Dependent Claim
131 words
The method of claim 1, wherein (i) each sequence-specific probe includes first and second probe elements having first and second oligonucleotides effective to bind to adjacent regions of a target sequence, where one of the oligonucleotides is derivatized with said polymer chain, (ii) said reacting is effective to bind both oligonucleotides to its specific target sequence, (iii) said treating includes ligating the oligonucleotides bound to the target polynucleotide under conditions which are effective to ligate the end subunits of target-bound oligonucleotides when their end subunits are base-paired with adjacent target bases, to form ligated probes, and releasing the ligated probe from the target polynucleotide, and (iv) the polymer chain attached to each different-sequence first oligonucleotide is effective to impart to the modified, labeled probe, a distinctive ratio of charge/translational frictional drag.
Claim 5
Dependent Claim
23 words
The method of claim 3, wherein the second probe element is reporter labeled, and said ligating is carried out with a ligase enzyme.
Claim 6
Dependent Claim
29 words
The method of claim 3, wherein said treating further includes subjecting the ligated probe to repeated cycles of probe binding and ligation, to amplify the concentration of ligated probe.
Claim 7
Dependent Claim
56 words
The method of claim 3, wherein said treating includes subjecting each ligated probe to repeated cycles of probe binding and ligation in the presence of a second pair of probe elements having oligonucleotides which, together, make up a sequence which is complementary to the selected ligated probe, to amplify the ligated probe in an exponential manner.
Claim 8
Dependent Claim
88 words
The method of claim 3, wherein said second probe element in each probe pair includes two alternative-sequence oligonucleotides which (i) are complementary to alternative sequences in the same portion of the associated target region and (ii) are derivatized with different detectable reporters, and said detecting includes determining the sequence of each of said regions according to (a) a signature electrophoretic migration rate of each probe, which identifies the target region associated with that probe, and (b) a signature reporter moiety, which identifies the mutation state of that region.
Claim 9
Dependent Claim
116 words
The method of claim 1, wherein (i) each sequence-specific probe includes first and second primer elements having first and second sequence-specific oligonucleotides effective to hybridize with opposite end regions of complementary strands of a target polynucleotide segment, respectively, where the oligonucleotide in the first primer element is derivatized with such probe-specific selected-polymer chain, (ii) said reacting is effective to bind both primer oligonucleotides to opposite end regions on complementary strands of the target polynucleotide, (iii) said treating is effective to amplify the target segment by primer-initiated polymerase chain reaction, and (iv) the polymer chain attached to each different-sequence first oligonucleotide is effective to impart to the amplified target sequences, a distinctive ratio of charge/translational frictional drag.
Claim 10
Dependent Claim
25 words
The method of claim 8, wherein the oligonucleotide in the second primer element is reporter labeled, and the labeled probes are double stranded polynucleotide fragments.
Claim 11
Dependent Claim
39 words
The method of claim 8, wherein said treating further includes hybridizing to the amplified target sequences, with such in single-stranded form, single-stranded, reporter-labeled oligonucleotides whose sequences are complementary to regions of the amplified target sequences, to form labeled probes.
Claim 12
Dependent Claim
69 words
The method of claim 1, wherein the binding polymers are oligonucleotides, each sequence-specific probe includes a binding polymer and an attached reporter label, the polymer chain associated with each different-sequence probe imparts to that probe, a distinctive ratio of charge/translational frictional drag, and said treating includes reacting the hybridized probes and target with DNA polymerase in the presence of a reporter-labeled nucleoside triphosphate molecule, to form said labeled probes.
Claim 13
Dependent Claim
78 words
The method of claim 1, wherein the binding polymers are oligonucleotides, each sequence-specific probe includes a binding polymer composed of first and second single-stranded DNA segments which mutually border a single-stranded RNA segment, where the polymer chain is attached to said first DNA segment, and a reporter is attached to said second DNA segment, and said treating includes reacting hybridized probe with an RNase enzyme specific for RNA/DNA substrate, to form modified, labeled probes lacking the polymer chains.
Claim 14
Dependent Claim
71 words
The method of claim 1, wherein the binding polymers are oligonucleotides, each sequence-specific probe includes a binding polymer composed of a first single-stranded DNA segment, and a second segment which includes single-stranded RNA, the polymer chain and reporter label are attached to said first segment, and said treating includes reacting hybridized probe with an RNase enzyme specific for RNA/DNA substrate, to form modified, labeled probes lacking said second binding polymer segments.
Claim 15
Dependent Claim
117 words
The method of claim 1, wherein the binding polymers are oligonucleotides, each sequence-specific probe includes (i) an oligonucleotide binding polymer having a 5' end, Where said polymer chain and a reporter label are attached to an oligonucleotide subunit adjacent said 5' end, and (ii) a primer designed for sequence-specific binding to the target upstream of said binding polymer, and said treating includes exposing immobilized probe to a DNA polymerase having a 5' to 3' exonuclease activity under conditions effective to extend said primer toward said binding polymer, said exposing being effective to enzymatically cleave said adjacent subunit from the binding polymer, forming a labeled probe whose polymer chain imparts to the probe, a distinctive charge/translational frictional drag.
Claim 16
Dependent Claim
76 words
The method of claim 1, wherein each sequence-specific probe includes a binding polymer and an attached reporter label, the polymer chain associated with each different-sequence probe imparts to that probe, a distinctive ratio of charge/translational frictional drag, and said treating includes immobilizing said target polynucleotide, washing the immobilized target polynucleotide to remove probes not bound to the target polynucleotide in a sequence-specific manner, and denaturing the target polynucleotide to release probes bound in a sequence-specific manner.
References Cited (Backward Citations)
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