US5118604A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Restriction-fragment DNA probes and probe clusters
Assignee: Yale University
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Filing: 1990-06-15
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Granted: 1992-06-02
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Expires: 2010-06-15
Abstract
Abstract A probe capable of binding by homologous base pairing independently to a pair of gene regions bordering the upstream and downstream sides of a pair of infrequent restriction endonuclease sites separated by between about 20-2,000 kilibases on a section of linear DNA. The probe is produced, according to the method of the invention by digesting the DNA section to completion with the selected endonuclease, and ligating the resulting fragments under conditions which favor end-to-end circularization, and selecting digest fragments of the large circular molecules which have end to end junctions. Also disclosed are method for mapping and ordering the positions of such probes, using another set of linking probes which span such rate cutting sites.
Inventors
- 👤 Sherman M. Weissman
- 👤 Francis Collins
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 06:51
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6841; C12Q1/6876
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
162 words
A probe comprising: a first DNA segment (1) derived from the upstream end region of a linear DNA fragment obtained by digestion of genomic DNA to completion with a restriction endonuclease which cuts at sites that occur infrequently, said DNA fragment having upstream and downstream end regions which are spaced from one another by between about 100 kilobases to 2,000 kilobases, 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 end region in genomic DNA; where the probe represents a subset of restriction fragments of said DNA, and the probe is of a size which can be cloned.
Claim 2
Dependent Claim
162 words
A probe comprising: a first DNA segment (1) derived from the upstream end region of a linear DNA fragment obtained by digestion of genomic DNA to completion with a restriction endonuclease which cuts at sites that occur infrequently, said DNA fragment having upstream and downstream end regions which are spaced from one another by between about 100 kilobases to 2,000 kilobases, 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 end region in genomic DNA; where the probe represents a subset of restriction fragments of said DNA, and the probe is of a size which can be cloned.
Claim 3
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 4
Dependent Claim
22 words
The probe of claim 1, wherein the intermediate segment is a selectable marker segment which allows probe selection in a cloning system.
Claim 5
Dependent Claim
29 words
The probe of claim 3, wherein the selectable marker segment is a suppressor tRNA which allows for selection of a phage vector containing the probe in suppressor minus host.
Claim 6
Dependent Claim
23 words
The probe of claim 2, wherein the intermediate segment is a ligand adapted to bind specifically and with high affinity to an anti-ligand.
Claim 7
Dependent Claim
18 words
The probe of claim 5, wherein the ligand is biotin, and the anti-ligand is avidin or anti-biotin antibody.
Claim 8
Dependent Claim
21 words
The probe of claim 1, wherein the restriction endonuclease is selected from the group consisting of NotI, SfiI, SalI, and MluI.
Claim 9
Dependent Claim
20 words
The probe of claim 1, wherein the upstream and downstream gene regions in the DNA fragment are each single-copy regions.
Claim 10
Dependent Claim
19 words
The probe of claim 1, wherein said first and second segments are connected by direct ligation to one another.
Claim 11
Independent Claim
192 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 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 100-2,000 kilobases, said method comprising: digesting the genomic DNA to completion with a restriction endonuclease which cuts at sites that occur infrequently to produce genomic fragments, where at least some of said fragments have 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 restriction endonuclease treatment to release digest fragments which are of a size which can be cloned, and which include fragments containing such connected fragment ends intact, 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 12
Independent Claim
192 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 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 100-2,000 kilobases, said method comprising: digesting the genomic DNA to completion with a restriction endonuclease which cuts at sites that occur infrequently to produce genomic fragments, where at least some of said fragments have 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 restriction endonuclease treatment to release digest fragments which are of a size which can be cloned, and which include fragments containing such connected fragment ends intact, 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 13
Dependent Claim
49 words
The method of claim 10, wherein said ligating is carried out in the presence of a selectable marker, which is adapted to join the cut ends of the genomic fragments, to produce circularized fragments each having one or more such selectable marker connecting the fragment ends to each other.
Claim 14
Independent Claim
312 words
A method of ordering restriction fragments along a DNA segment comprising: providing a pool of hopping probes representative of the DNA segment, each hopping probe containing a first DNA segment (1) derived from the upstream end region of a linear DNA fragment obtained by digestion of the DNA segment to completion with a restriction endonuclease which cuts at sites that occur infrequently, said DNA fragment having upstream and downstream end regions which are spaced from one another by between about 100 kilobases to 2,000 kilobases, 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 end region in genomic DNA; where the probe represents a subset of restriction fragments of said DNA, and the probe is of a size which can be cloned; providing a pool of linking probes representative of the DNA segment, obtained by digesting the DNA segment with a restriction endonuclease whose cut site does not overlap with the cut site of the restriction enzyme used to generate the hopping probes; and, alternately hybridizing a selected linking probe to the pool of hopping probes to identify a pair of hopping probes which have regions of homologous overlap with the selected linking probe, and then hybridizing one of the identified hopping probes with the pool of linking probes, to identify a pair of linking probes which have homologous regions of overlap with the one hopping probe, at each hybridization step, identifying the next-in-sequence hopping or linking probe, and using the next-in-sequence probe in the next hybridization step.
Claim 15
Independent Claim
312 words
A method of ordering restriction fragments along a DNA segment comprising: providing a pool of hopping probes representative of the DNA segment, each hopping probe containing a first DNA segment (1) derived from the upstream end region of a linear DNA fragment obtained by digestion of the DNA segment to completion with a restriction endonuclease which cuts at sites that occur infrequently, said DNA fragment having upstream and downstream end regions which are spaced from one another by between about 100 kilobases to 2,000 kilobases, 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 end region in genomic DNA; where the probe represents a subset of restriction fragments of said DNA, and the probe is of a size which can be cloned; providing a pool of linking probes representative of the DNA segment, obtained by digesting the DNA segment with a restriction endonuclease whose cut site does not overlap with the cut site of the restriction enzyme used to generate the hopping probes; and, alternately hybridizing a selected linking probe to the pool of hopping probes to identify a pair of hopping probes which have regions of homologous overlap with the selected linking probe, and then hybridizing one of the identified hopping probes with the pool of linking probes, to identify a pair of linking probes which have homologous regions of overlap with the one hopping probe, at each hybridization step, identifying the next-in-sequence hopping or linking probe, and using the next-in-sequence probe in the next hybridization step.
Claim 16
Dependent Claim
29 words
The hopping probe of claim 12, wherein the restriction endonuclease which cuts at sites that occur infrequently is selected from the group consisting of NotI, SfiI, SalI, and MluI.
Claim 17
Dependent Claim
56 words
The method of claim 12, wherein providing the pool of linking probes includes the steps of digesting the DNA segment with a restriction endonuclease which cleaves the segment into fragments of about 20 kilobases or less, and isolating those fragments which contain an internal cut site of the restriction enzyme used to generate the hopping probes.
Claim 18
Dependent Claim
52 words
The method of claim 14, wherein said isolating includes cloning the fragments in a circular cloning vector, linearizing the vectors with an endonuclease which cuts at the internal cut site of the restriction enzyme used to one or both ends of the linearized vector, and isolating those vectors containing the binding sequence.
Claim 19
Dependent Claim
34 words
The method of claim 15, wherein the binding sequence is biotinylated, and said isolating is based on specific binding of the linearized, biotin-containing vectors to a solid support having attached avidin or antibiotin antibody.
Claim 20
Dependent Claim
57 words
The method of claim 14, wherein said isolating includes cloning the fragments in a circular cloning vector, linearizing the vector with a restriction endonuclease which cuts specifically at the internal cut site of the restriction enzyme used to generate the hopping probes, introducing a selectable marker into the linearized vectors, and selecting vectors containing the selectable marker.
Claim 21
Dependent Claim
29 words
The method of claim 17, wherein the selectable marker is a suppressor tRNA and the vectors are selected on the basis of growth in a suppressor minus bacterial host.
Claim 22
Dependent Claim
63 words
The method of claim 14, wherein said isolating includes circularizing the digest fragments in the presence of a selectable marker, digesting the circularized fragments with a restriction endonuclease which cuts specifically at the internal cut site of the restriction enzyme used to generate the hopping probes, introducing the cut vectors into a cloning vector, and selecting cloning vectors which contain the selectable marker.
Claim 23
Dependent Claim
214 words
A family of sequence-overlapping probes derived from a segment of genomic DNA comprising: a first DNA segment (1) derived from the upstream end region of a linear DNA fragment obtained by digestion of the DNA segment to completion with a restriction endonuclease which cuts at sites that occur infrequently, said DNA fragment having upstream and downstream end regions which are spaced from one another by between about 100 kilobases to 2,000 kilobases, 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 end region in genomic DNA, where the probe represents a subset of restriction fragments of said DNA, and the probe is of a size which can be cloned; and, a pool of linking probes representative of the DNA segment, obtained by digesting the DNA segment with a restriction endonuclease whose cut site does not overlap with the cut site of the restriction enzyme used to generate the hopping probes.
Claim 24
Dependent Claim
214 words
A family of sequence-overlapping probes derived from a segment of genomic DNA comprising: a first DNA segment (1) derived from the upstream end region of a linear DNA fragment obtained by digestion of the DNA segment to completion with a restriction endonuclease which cuts at sites that occur infrequently, said DNA fragment having upstream and downstream end regions which are spaced from one another by between about 100 kilobases to 2,000 kilobases, 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 end region in genomic DNA, where the probe represents a subset of restriction fragments of said DNA, and the probe is of a size which can be cloned; and, a pool of linking probes representative of the DNA segment, obtained by digesting the DNA segment with a restriction endonuclease whose cut site does not overlap with the cut site of the restriction enzyme used to generate the hopping probes.
References Cited (Backward Citations)
US4710465A
Cited
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