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US 6,258,571 B1Granted
High throughput DNA sequencing vector
AssigneeGenset S.A.
InventorsIlya Chumakov, Hiroaki Tanaka
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Abstract
High throughput DNA sequencing vectors for generating nested deletions using enzymatic techniques and/or transposition-based techniques are disclosed. Methods of constructing contigs of long DNA sequences and methods of generating nested deletions are also disclosed. A truncated lacZ derivative useful in measuring the copy number of the lacZ derivative in a host cell is also disclosed.
Claims26
A method of generating a deletion in an insert comprising:
- (a) providing a vector comprising at least one cloning site and transposition elements positioned on each side of said at least one cloning site, said transposition elements adapted for generating deletions in said insert;
- (b) cloning an insert into said at least one cloning site of vector;
- (c) introducing said vector into a host cell containing a nucleic acid encoding a transposase, wherein said vector containing an insert is maintained at a low copy number in said host cell;
- and
- (d) inducing transposase activity in said host cell, thereby mediating transposition into said insert.
2 more elements
A method of constructing a contig containing the complete sequence of a piece of DNA comprising:
- (a) subcloning fragments of said piece of DNA into a vector comprising at least one cloning site and transposition elements positioned on each side of said at least one cloning site, said transposition elements being adapted for generating a deletion in said subcloned fragments, wherein said vectors containing said subcloned fragments are maintained at low copy number in host cells;
- (b) obtaining the complete sequence of a portion of said subcloned fragments;
- (c) obtaining the sequence of the ends of the remainder of said subcloned fragments;
- (d) identifying overlapping subclones to construct one or more scaffolds covering the complete sequence of said piece of DNA;
- (e) introducing said vectors containing at least some of said overlapping subclones into host cells which comprise a nucleic acid encoding a transposase and inducing transposase activity in said host cells, thereby generating deletions in said overlapping subclones;
- and
- (f) completely sequencing said deletions.
4 more elements
Dependencies are inferred from the claim text.