Method for generation of sequence sampled maps of complex genomes
AssigneeThe Salk Institute for Biological Studies
InventorsGlen A. Evans, Michael W. Smith
Life of this patent
Estimating the term…
Abstract
The present invention relates to a rapid and powerful sequence "sequence sampled mapping" method for sequencing complex genomes. The invention method is applicable to genomic DNA, preferably mammalian chromosomes, and in a preferred embodiment, employs a "bottom-up" mapping strategy, which allows for the simultaneous analysis of multiple cosmid clones for the detection of overlaps. The sequence sample mapping method is useful first, for the completion of high density sequence-based maps, and ultimately, for the complete sequencing of genomic DNA directly from cosmid clones.
Claims20
A method for sequencing complex genomes, said method comprising:
- (1) sequencing at least 100 nucleotides from the end of each member of a library of cosmid clones,
- wherein said cosmid clones are prepared by inserting genomic DNA fragments into cosmid vectors, and
- 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 nucleic acids specific to the ends of the inserted DNA,
- (2) determining the relative spatial relationship between the cosmid clones, and
- (3) assembling a sequence sampled map by correlating the end-specific nucleotide sequence information with the relative spatial relationship between the cosmids.
A method for sequencing complex genomes, said method comprising:
- (1) 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,
- (2) arranging the cosmid clones, whereby each clone may be identified and replicas of said arrangement may be reproduced,
- (3) pooling portions of said 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,
- (4) 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,
- (5) identifying the cosmid clones from among those identified in step (4) that hybridize to two or more pools of probes, thereby identifying groups of cosmid clones that include overlapping DNA,
- (6) assembling contigs from said groups, and
- (7) sequencing the fragment ends of the DNA inserts of each of the overlapping cosmid clones.
Dependencies are inferred from the claim text.