High throughput DNA sequencing vector
View Patent ↗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.
1. A method for identifying vectors containing a DNA insert, comprising the steps of:
subjecting a plurality of vectors to insertion conditions under which an insert is placed in at least some of said vectors, wherein said vectors have a first copy number if no insert occurs and a second copy number if an insert occurs; and
identifying at least some vectors containing an insert by screening said vectors for said second copy number.
2. The method of claim 1 , wherein said vectors comprise hybridization sites for insert sequencing primers, said hybridization sites for insert sequencing primers being positioned so as to allow the sequencing of said DNA insert.
3. The method of claim 1 , wherein said vectors comprise hybridization sites for insert amplifying primers, said hybridization sites for insert amplifying primers being positioned so as to allow the amplification of said DNA insert.
4. The method of claim 1 , wherein said vectors comprise a single stranded origin of replication, said single stranded origin of replication permitting the isolation of said vector in a single stranded form.
5. The method of claim 1 , wherein said vectors containing a DNA insert are present at a low copy number.
6. The method of claim 1 , wherein said first copy number is a high copy number and said second copy number is a low copy number.
7. The method of claim 1 , wherein said vectors comprise at least one copy number indicator for indicating the copy number of said vectors in cells.
8. The method of claim 7 , wherein said copy number indicator comprises a selectable marker.
9. The method of claim 1 , wherein said screening step comprises determining the copy number of a vector.
10. A method to determine the copy number of a vector comprising a truncated lacZ gene, said method comprising the steps of:
introducing said vector into a host cell, wherein said truncated lacZ gene, when present at a high copy number in said host cells, confers dark blue coloration to said host cells grown on a medium containing Xgal and IPTG, and wherein said truncated lacZ gene, when present at a low copy number in said host cells, confers light blue coloration to said host cells grown on said medium; and
determining the color of the host cells when the host cells are grown on said medium.
11. A method to determine the copy number of a vector comprising the strA+ gene, said method comprising the steps of:
introducing said vector into a streptomycin resistant host cell, wherein said host cells are unable to grow in the presence of streptomycin when the strA gene is present at a high copy number and wherein said host cells are able to grow in the presence of streptomycin when the strA gene is present at a low copy number; and
determining the ability of said host cells to grow on a medium containing streptomycin.
12. The method according to claim 11 , wherein said vector further comprises a high copy number origin of replication and a low copy number origin of replication.
13. The method according to claim 12 , wherein said high copy number origin of replication contains one or more cloning sites.
14. The method according to claim 11 , wherein said vector comprises additional selectable markers.
15. The method according to claim 10 , wherein said truncated LacZ gene comprises SEQ ID NO: 7.
16. The method according to claim 15 , wherein said truncated LacZ gene comprises nucleotides 1-292 of SEQ ID NO: 7.
17. The method according to claim 10 , wherein said truncated LacZ gene comprises nucleotides 1-392 of the LacZ gene sequence.