Restriction-fragment DNA probes and probe clusters
First Claim
1. 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.
0 Assignments
0 Petitions
Accused Products
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.
-
Citations
20 Claims
-
1. 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
-
-
10. 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. - View Dependent Claims (11)
-
-
12. 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. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
- providing a pool of hopping probes representative of the DNA segment, each hopping probe containing
-
20. 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.
-
Specification