US5219726A
Expired / Lapsed
US
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Physical mapping of complex genomes
Assignee: Salk Institute for Biological Studies
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Filing: 1989-06-02
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Granted: 1993-06-15
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Expires: 2009-06-02
Abstract
Abstract Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.
Inventors
- 👤 Glen A. Evans
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 20:38
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12N; C12N15/00; C12N15/09; C12N15/10; C12Q1/6834; C12Q1/6837; C12Q1/6841; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
174 words
A method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones, comprising: (a) arranging the multiple cosmid clones, whereby each clone may be identified and replicas of said arrangement may be generated; (b) pooling a first portion of the multiple cosmid clones and synthesizing mixed end-specific RNA probes from the DNA inserts that have been prepared from said pooled clones, wherein said portion includes less than all of said multiple cosmid clones; (c) hybridizing the probes to a replica of said arranged cosmid clones and identifying the cosmid clones in the replica that hybridize to the probes, wherein said identified clones include the pooled cosmid clones and cosmid clones that contain DNA inserts that overlap with the DNA inserts in the pooled clones; (d) repeating said hybridization step with a second portion of mixed end-specific probes that are prepared from a second pooled portion of multiple cosmid clones; and (e) identifying the cosmid clones in each replica to which both the probes of steps b) and d) hybridize thereby identifying overlapping clones.
Claim 2
Independent Claim
174 words
A method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones, comprising: (a) arranging the multiple cosmid clones, whereby each clone may be identified and replicas of said arrangement may be generated; (b) pooling a first portion of the multiple cosmid clones and synthesizing mixed end-specific RNA probes from the DNA inserts that have been prepared from said pooled clones, wherein said portion includes less than all of said multiple cosmid clones; (c) hybridizing the probes to a replica of said arranged cosmid clones and identifying the cosmid clones in the replica that hybridize to the probes, wherein said identified clones include the pooled cosmid clones and cosmid clones that contain DNA inserts that overlap with the DNA inserts in the pooled clones; (d) repeating said hybridization step with a second portion of mixed end-specific probes that are prepared from a second pooled portion of multiple cosmid clones; and (e) identifying the cosmid clones in each replica to which both the probes of steps b) and d) hybridize thereby identifying overlapping clones.
Claim 3
Dependent Claim
21 words
The method of claim 1, wherein said second portion includes at least one clone that was present in the first portion.
Claim 4
Dependent Claim
63 words
The method of claim 1, wherein one clone from said first portion is added to said second portion of pooled clones prior to preparing probes therefrom, whereby the clones that hybridize to both portions of probes, other than those that include the DNA inserts from which the probes were synthesized, contain DNA inserts that overlap with said added clone from the first portion.
Claim 5
Dependent Claim
95 words
The method of claim 1, further comprising: (f) repeating said hybridization step with an additional portion of mixed end-specific probes that are prepared from an additional portion of the pooled multiple clones, wherein said additional portion includes at least one clone that was present in the first and second portions, but does not include any other clones that were previously pooled; and (g) identifying the cosmid clones in the library to which the probes of steps b), e), and f) hybridize, wherein the identified clones are other than those which correspond to the pooled clones.
Claim 6
Dependent Claim
34 words
The method of claim 4, further comprising: (h) repeating steps (f) and (g) a plurality of times until all of the cosmid clones in the library have been pooled and hybridized to the library.
Claim 7
Dependent Claim
28 words
The method of claim 1, wherein the arrangement is a two-dimensional matrix and the clones are pooled pairwise according to the rows and columns of a two-dimensional matrix.
Claim 8
Dependent Claim
39 words
The method of claim 1, wherein the arrangement is a three-dimensional matrix and the clones are pooled according to intersecting planes of the three-dimensional matrix, wherein following hybridization the replicas are compared according to intersecting planes of the matrix.
Claim 9
Dependent Claim
58 words
The method of claim 7, wherein groups of three replicas produced by hybridizing probes prepared from pooled clones according to three intersecting planes are compared, whereby the clones on all three replicas that hybridize to probes from each of the pooled clones include DNA that overlaps with the clone that occurs at the intersection of the three planes.
Claim 10
Dependent Claim
33 words
The method of claim 1, wherein said cosmid clones include sequence of nucleotides flanking at least one end of the inserted DNA that serve as promoters for the synthesis of the end-specific probes.
Claim 11
Dependent Claim
41 words
The method of claim 9, wherein said at least one of the flanking sequences includes a sequence of nucleotides that is recognized as a promoter by a bacteriophage polymerase, and that is positioned operatively for transcription of the inserted DNA fragment.
Claim 12
Dependent Claim
39 words
The method of claim 10, wherein both flanking sequences include sequences of nucleotides that are recognized as promoters by a bacteriophage RNA polymerase, wherein said promoters are oppositely oriented and positioned operatively for transcription of the inserted DNA fragment.
Claim 13
Dependent Claim
35 words
The method of claim 11, wherein each of the bacteriophage RNA polymerase-specific promoters is selected from the group consisting of promoters specific for bacteriophage T7 RNA polymerase, and promoters specific for bacteriophage T3 RNA polymerase.
Claim 14
Dependent Claim
34 words
The method of claim 9, wherein said cosmid clones are prepared by inserting DNA fragments into the cloning sites of a cosmid vector selected from the group consisting of pWE8, pWE10, pWE15, and PWE16.
Claim 15
Independent Claim
20 words
The method of any one of claims 10 through 12, wherein said cosmid clones include at least two cos sites.
Claim 16
Dependent Claim
33 words
The method of claim 9, wherein said cosmid clones are prepared by inserting DNA fragments into the cloning sites of a cosmid vector selected from the group consisting of sCOS-1, sCOS-2, and sCOS-4.
Claim 17
Independent Claim
238 words
A method for physical mapping of complex genomes comprising: (a) preparing a genomic library of cosmid clones by inserting DNA fragments from said genome into cosmid vectors, wherein the cosmid vectors include sequences of nucleotides that flank at least one end of the inserted DNA and that serve as transcription initiation sites for the synthesis of end-specific probes; (b) arranging the cosmid clones, whereby each clone may be identified and replicas of said arrangement may be generated; (c) pooling portions of cosmid clones and synthesizing pools of mixed end-specific probes from the DNA inserts that have been prepared from said pooled clones, wherein each pool contains fewer than all of the cosmid clones in the library but all of the cosmid clones in the library are included in at least one pool; (d) hybridizing each pool of probes to a replica of said arranged cosmid clones and identifying the cosmid clones in each replica that hybridize to the probes, wherein said identified clones include the pooled cosmid clones and cosmid clones that contain DNA inserts that overlap with the DNA inserts in the pooled clones; (e) identifying the cosmid clones from among those identified in step (d) the clones that hybridize to two or more pools of probes, thereby identifying groups of cosmid clones that include overlapping DNA; and (g) assembling contigs from said groups into a physical map of the genome from which the library was derived.
Claim 18
Independent Claim
238 words
A method for physical mapping of complex genomes comprising: (a) preparing a genomic library of cosmid clones by inserting DNA fragments from said genome into cosmid vectors, wherein the cosmid vectors include sequences of nucleotides that flank at least one end of the inserted DNA and that serve as transcription initiation sites for the synthesis of end-specific probes; (b) arranging the cosmid clones, whereby each clone may be identified and replicas of said arrangement may be generated; (c) pooling portions of cosmid clones and synthesizing pools of mixed end-specific probes from the DNA inserts that have been prepared from said pooled clones, wherein each pool contains fewer than all of the cosmid clones in the library but all of the cosmid clones in the library are included in at least one pool; (d) hybridizing each pool of probes to a replica of said arranged cosmid clones and identifying the cosmid clones in each replica that hybridize to the probes, wherein said identified clones include the pooled cosmid clones and cosmid clones that contain DNA inserts that overlap with the DNA inserts in the pooled clones; (e) identifying the cosmid clones from among those identified in step (d) the clones that hybridize to two or more pools of probes, thereby identifying groups of cosmid clones that include overlapping DNA; and (g) assembling contigs from said groups into a physical map of the genome from which the library was derived.
Claim 19
Dependent Claim
39 words
The method of claim 16, wherein each portion includes at least one common clone that was present in one of the other portions, whereby the clones identified in step (e) contain DNA inserts that overlap with the common clone.
Claim 20
Dependent Claim
42 words
The method of claim 16, wherein in step (e) the cosmid clones in each replica that include clones that hybridize to two or more pools are identified by comparing pairs of replicas produced by hybridizing pools that include one clone in common.
Claim 21
Dependent Claim
30 words
The method of claim 16, wherein the location of each individual clone in the replica is identified by unique coordinates that describe the location of the clone in the replica.
Claim 22
Dependent Claim
28 words
The method of claim 16, wherein the arrangement is a matrix, and the location of each clone int he matrix is uniquely identified by at least two coordinates.
Claim 23
Dependent Claim
27 words
The method of claim 20, wherein the clones whose locations include one or more common coordinates and at least one different coordinate are pooled in step (c).
Claim 24
Dependent Claim
46 words
The method of claim 16, wherein said cosmid vectors contain at least one promoter specific for a bacteriophage RNA polymerase and a cloning site for the insertion of DNA fragments, wherein aid promoter is positioned operatively for transcription of a DNA fragment into said cloning site.
Claim 25
Dependent Claim
39 words
The method of claim 22, wherein said cosmid vectors contain two oppositely oriented promoters, each of which is specific for a bacteriophage RNA polymerase and is positioned operatively for transcription of a DNA fragment inserted into said cloning site.
Claim 26
Dependent Claim
35 words
The method of claim 23, wherein each of said bacteriophage RNA polymerase-specific promoters is selected from the group consisting of promoters specific for bacteriophage T7 RNA polymerase, and promoters specific for bacteriophage T3 RNA polymerase.
Claim 27
Dependent Claim
21 words
The method of claim 24, wherein said cosmid vector is selected from the group consisting of pWE8, pWE10, pWE15, and pWE16.
Claim 28
Independent Claim
20 words
The method of any one of claims 17 through 20, wherein said cosmid vectors contain at least two cos sites.
Claim 29
Dependent Claim
15 words
The method of claim 26, wherein said cos sites are separated by unique restriction sites.
Claim 30
Dependent Claim
20 words
The method of claim 27, wherein said cosmid vector is selected from the group consisting of sCOS-1, sCOS-2, and sCOS-4.
References Cited (Backward Citations)
US4766072A
Cited
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