Physical mapping of complex genomes
First Claim
1. 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.
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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.
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Citations
28 Claims
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1. A method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones, comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for physical mapping of complex genomes comprising:
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(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. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification