US4710465A
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Junction-fragment DNA probes and probe clusters
Assignee: Yale University
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Filing: 1984-04-19
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Granted: 1987-12-01
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Expires: 2004-04-19
Abstract
Abstract A junction-fragment DNA probe, a DNA probe cluster, and methods of preparing and using the probe and cluster to study gene localization and organization. The probe includes first and second gene segments which are derived from first and second, single-copy genomic-DNA gene regions, respectively, separated from one another, in the genomic DNA strand, by a selected distance of between about 20 and 2,000 kilobases. The two segments in the probe are connected at a j The invention was supported in part by the National Institute of Health grant #NIH CA-30938 and the Government has certain rights to the invention.
Inventors
- 👤 Sherman M. Weissman
- 👤 Francis Collins
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 03:33
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6841; C12Q1/6876; Y; Y10; Y10S; Y10S436/00; Y10S436/811
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
187 words
A method of forming a probe capable of binding by homologous base pairing independently to an upstream gene region which is present on a linear section of mammalian genomic DNA and which binds by homologous base pairing to a selected probe, and to a downstream gene region which is also present on said section and is spaced from the upstream gene region by a distance of about 20-2,000 kilobases, said method comprising digesting the mammalian genomic DNA section by endonuclease treatment to produce genomic fragments, at least some of which have sizes consistent with section fragments containing said upstream and downstream regions at their opposite ends, ligating the genomic fragments under fragment concentration conditions which favor circularization of single fragments into circular DNA species with connected fragment ends, digesting the circular DNA species by endonuclease treatment to release digest fragments which are of a size which can be cloned, and which include fragments containing such connected fragment ends, cloning the digest fragments, and isolating cloned digest fragments which contain the connected fragment ends, and which are able to bind by homologous base pairing to the selected probe.
Claim 2
Independent Claim
187 words
A method of forming a probe capable of binding by homologous base pairing independently to an upstream gene region which is present on a linear section of mammalian genomic DNA and which binds by homologous base pairing to a selected probe, and to a downstream gene region which is also present on said section and is spaced from the upstream gene region by a distance of about 20-2,000 kilobases, said method comprising digesting the mammalian genomic DNA section by endonuclease treatment to produce genomic fragments, at least some of which have sizes consistent with section fragments containing said upstream and downstream regions at their opposite ends, ligating the genomic fragments under fragment concentration conditions which favor circularization of single fragments into circular DNA species with connected fragment ends, digesting the circular DNA species by endonuclease treatment to release digest fragments which are of a size which can be cloned, and which include fragments containing such connected fragment ends, cloning the digest fragments, and isolating cloned digest fragments which contain the connected fragment ends, and which are able to bind by homologous base pairing to the selected probe.
Claim 3
Dependent Claim
67 words
The method of claim 1, for producing a probe capable of binding by homologous base pairing to such upstream and downstream gene regions, where such are separated by a selected distance between about 20 and 2,000 kilobases, which further includes, prior to said ligating, size fractionating the genomic fragments and selecting for ligating, those fragments having sizes corresponding to the selected distance between the two gene regions.
Claim 4
Dependent Claim
89 words
The method of claim 1, for producing a second probe capable of binding independently to the downstream gene region and to a third gene region present on the linear DNA section, which is spaced from and downstream fo the downstream gene region by a distance between about 20 and 2,000 kilobases, which further includes isolating cloned digest fragments which contain the connected fragment ends, and which are able to bind by homologous base pairing to the probe isolated by the first isolating step, but not to such selected probe.
Claim 5
Dependent Claim
85 words
The method of claim 1, for producing a cluster of probes, all of which are capable of binding independently by homologous base pairing to such upstream gene region, and each of which is capable of binding by homologous base pairing to one of a series of such downstream regions which are separated from the upstream gene region by different distances between about 20 and 2,000 kilobases, wherein said digesting is carried out under conditions which produce fragments having heterogeneous sizes greater than about 20 kilobases.
Claim 6
Dependent Claim
50 words
The method of claim 4, for use in localizing such upstream gene region on the linear DNA strand, which further includes labeling the cluster of probes with a reporter, hybridizing the cluster of labelled probes with the linear DNA strand, and identifying the region of DNA section having bound reporter.
Claim 7
Dependent Claim
59 words
The method of claim 1, which further cleaving the isolated digest fragments which contain the connected fragment ends, and which are able to bind by homologous base pairing to the selected probe, to release digest fragment portions which are able to bind by homologous base pairing to the downstream, but not the upstream gene region of the DNA section.
Claim 8
Dependent Claim
61 words
The method of claim 1, wherein said ligating is performed within a concentration range of genomic fragments which is substantially inversely proportional to the square root of the sizes of the fragment where the ligating is performed in a concentration range of between about 0.5 to 1.0 micrograms fragment DNA per ml for fragments in the approximately 50 kilobase size range.
Claim 9
Dependent Claim
37 words
The method of claim 1, which further includes, prior to said ligating, attaching to the DNA genomic fragments, a selectable marker by which a released digest fragment containing such marker can be selected in a cloning system.
Claim 10
Dependent Claim
36 words
The method of claim 8, wherein the marker includes a suppressor tRNA gene, for selection in a cloning system which includes a phage vector carrying such fragment, and a bacterial vector host which is suppressor-tRNA negative.
Claim 11
Dependent Claim
44 words
The method of claim 1, which further includes, prior to said ligating, attaching to the DNA genomic fragments, a ligand marker segment by which a released monomer fragment containing the marker segment can be isolated by binding to an anti-ligand, prior to said cloning.
Claim 12
Dependent Claim
22 words
The method of claim 1, wherein the ligand marker is a biotinylated DNA segment, and the anti-ligand is avidin or anti-biotin antibody.
Claim 13
Dependent Claim
141 words
A probe comprising a first DNA segment (1) derived from the upstream end region of a linear fragment of mammalian genomic DNA having upstream and downstream end regions which are spaced from one another by between about 20-2,000 kilobase and (2) which is effective to bind by homologous base pairing to said upstream end region in genomic DNA, and a second DNA segment (1) derived from the downstream end region of said DNA fragment and connected adjacent its downstream end, as defined by the upstream-to-downstream orientation in the DNA fragment, to the upstream end of the first segment, and (2) which is effective to bind by homologous base pairing to said downstream and region in genomic DNA, where the total length of the probe is substantially less than that of said DNA fragment, and of a size which can be cloned.
Claim 14
Dependent Claim
141 words
A probe comprising a first DNA segment (1) derived from the upstream end region of a linear fragment of mammalian genomic DNA having upstream and downstream end regions which are spaced from one another by between about 20-2,000 kilobase and (2) which is effective to bind by homologous base pairing to said upstream end region in genomic DNA, and a second DNA segment (1) derived from the downstream end region of said DNA fragment and connected adjacent its downstream end, as defined by the upstream-to-downstream orientation in the DNA fragment, to the upstream end of the first segment, and (2) which is effective to bind by homologous base pairing to said downstream and region in genomic DNA, where the total length of the probe is substantially less than that of said DNA fragment, and of a size which can be cloned.
Claim 15
Dependent Claim
25 words
The probe of claim 1, wherein the first and second segments are joined through an intermediate segment which is not derived from said DNA fragment.
Claim 16
Dependent Claim
22 words
The probe of claim 13, wherein the intermediate segment is a selectable marker segment which allows probe selection in a cloning system.
Claim 17
Dependent Claim
30 words
The probe of claim 14, wherein the selectable marker segment is a suppressor tRNA which allows for selection of a phage vector containing the probe in a suppressor (-) host.
Claim 18
Dependent Claim
23 words
The probe of claim 13, wherein the intermediate segment is a ligand adapted to bind specifically and with high affinity to an anti-ligand.
Claim 19
Dependent Claim
18 words
The probe of claim 16, wherein the ligand is biotin, and the anti-ligand is avidin or anti-biotin antibody.
Claim 20
Dependent Claim
50 words
The probe of claim 12, for use in binding to upstream and downstream gene regions which are spaced a seelcted distance from one another in the DNA section, wherein the DNA fragment from which said first and second segments are derived has a length substantially equal to such selected distance.
Claim 21
Dependent Claim
20 words
The probe of claim 12, wherein the upstream and downstream gene regions in the DNA fragment are each single-copy regions.
Claim 22
Dependent Claim
19 words
The probe of claim 12, wherein said first and second segments are connected by direct ligation to one another.
Claim 23
Dependent Claim
177 words
A cluster of probes each comprising a first DNA segment (1) derived from the upstream end region of one in a group of fragments of mammalian genomic DNA, where each fragment in the group has upstream and downstream end regions which are spaced from one another by between about 20-2,00 kilobases, and where the fragments in the group have the same upstream end region and different downstream end regions, and (2) which is effective to bind by homologous base pairing to said one fragment's upstream end region in genomic DNA, and a second DNA segment (1) derived from the downstream end region of said one DNA fragment and connected adjacent its downstream end, as defined by the upstream-to-downstream orientation in the DNA fragment, to the upstream end of the first segment, and (2) which is effective to bind by homologous base pairing to said one fragment's downstream end region in genomic DNA, where the total length of the probe is substantially less than that of said one DNA fragment, and of a size which can be cloned.
Claim 24
Dependent Claim
177 words
A cluster of probes each comprising a first DNA segment (1) derived from the upstream end region of one in a group of fragments of mammalian genomic DNA, where each fragment in the group has upstream and downstream end regions which are spaced from one another by between about 20-2,00 kilobases, and where the fragments in the group have the same upstream end region and different downstream end regions, and (2) which is effective to bind by homologous base pairing to said one fragment's upstream end region in genomic DNA, and a second DNA segment (1) derived from the downstream end region of said one DNA fragment and connected adjacent its downstream end, as defined by the upstream-to-downstream orientation in the DNA fragment, to the upstream end of the first segment, and (2) which is effective to bind by homologous base pairing to said one fragment's downstream end region in genomic DNA, where the total length of the probe is substantially less than that of said one DNA fragment, and of a size which can be cloned.
Claim 25
Dependent Claim
22 words
The cluster of claim 21, wherein the second segment in each probe is derived from a single-copy gene of said DNA fragment.
Claim 26
Dependent Claim
186 words
A cluster of probes each comprising a first DNA segment (1) derived from the upstream end region of one in a group of fragments of mammalian genomic DNA, where each fragment in the group has upstream and downstream end regions which are spaced from one another by between about 20-2,000 kilobases, and where the downstream end region of one fragment in the group is essentially the same as the upstream end region of another fragment in the group, and (2) which is effective to bind by homologous base pairing to said one fragment's upstream end region in genomic DNA, and a second DNA segment (1) derived from the downstream end region of said one DNA fragment and connected adjacent its downstream end, as defined by the upstream-to-downstream orientation in the DNA fragment, to the upstream end of the first segment, and (2) which is effective to bind by homologous base pairing to said one fragment's downstream end region in genomic DNA, where the total length of the probe is substantially less than that of said one DNA fragment, and of a size which can be cloned.
Claim 27
Dependent Claim
186 words
A cluster of probes each comprising a first DNA segment (1) derived from the upstream end region of one in a group of fragments of mammalian genomic DNA, where each fragment in the group has upstream and downstream end regions which are spaced from one another by between about 20-2,000 kilobases, and where the downstream end region of one fragment in the group is essentially the same as the upstream end region of another fragment in the group, and (2) which is effective to bind by homologous base pairing to said one fragment's upstream end region in genomic DNA, and a second DNA segment (1) derived from the downstream end region of said one DNA fragment and connected adjacent its downstream end, as defined by the upstream-to-downstream orientation in the DNA fragment, to the upstream end of the first segment, and (2) which is effective to bind by homologous base pairing to said one fragment's downstream end region in genomic DNA, where the total length of the probe is substantially less than that of said one DNA fragment, and of a size which can be cloned.
Claim 28
Dependent Claim
28 words
The cluster of claim 23, wherein the first and second segments in each probe are joined through an intermediate segment which is not derived from the DNA fragment.
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) | 1990-11-30 | 1991-06-01 | 1991-12-01 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-11-30 | 1995-06-01 | 1995-12-01 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-11-30 | 1999-06-01 | 1999-12-01 | $7700.00 | Window Closed / Lapsed |