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Nucleic Acids Research
Volume 17 Number 22 1989
pBluescript II: gene mapping vectors
M.A.Alting-Mees* and J.M.Short
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037, USA
Submitted October 9, 1989
Four new muitipurpose phagemid vectors, pBluescriptll SK+, pBluescriptll SK-, pBluescriptll
KS+ and pBkjescriptil KS-, varying in the orientation of their polylinker (KS versus SK) and fl origin (+
versus -) have been generated. These vectors were designed to facilitate rapid mapping of DNA inserts.
The mapping technique, based on a strategy described by Wahl at at (ref. 1), requires the presence of
unique restriction sites flanking both the DNA insert arnd two DNA hybridization target sequences. In
order to utilize this approach, BssH!l sites were insedted outside the T3 and T7 promoters of the
pBluescript phagemids (ref.2) (see Figure a). In order to obtain the map of a DNA insert, complete
digestion of the clone with BssHlI is followed by partial digestion with the mapping enzyme of choice. The
digests are analy7od by Southern blot technique hybridizing with T3 or T7 oligonucleotide probes to
determine the location of each restriction enzyme site relative to the BssHII site (ref.3)(see Figure b for a
graphic representation of this method).
Additional features of the pBluescript 11 vectors are common to the original pBluescript
phagemids (ref.2) and include: (1) Single strand DNA rescue of either strand (using the two orientations
of the fI origin) wih the aid of filamentous helper phage. (2) 21 unique restriction enzyme sites in the
polylinker in either orientation relative to the f-gal promoter. (3) The arrangement of restriction sites
within the polylinker allowing unidirectional deletions by the exonuclease III/mung bean nuclease
technique (ref.4,5). (4) Biue/white colour selection of recombinant clones and production of ,8-gal fusion
proteins by IPTG induicible transcription from the 3-gal promoter through the polylinker and amino terminal
portion of the f-gal gene. (5) 13 and T7 RNA promoters for production of high levels of sense or antisense RNA transcripts of.the cloned insert (ref.6,7).
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Figure b. Mapping Strategy
*To whom correspondence should be addressed
References
1. Wahl, G.M., Lawis, K.A., Ruiz, J.C., Rothenberg, B., Zhao, J. and Evans, G.A., (1987) Proc. Nad. Acad. Sci. USA
84:2160-2164. 2. Short, J.M., Fernandez, J.M., Sorge, J.A., and Huse, W.D., (1988) Nucl. Acids Res. 16(15):7583-7600. 3. Pita,
A. (1989) Strategies, 2(4). 4. Guo, IL.H., Yang, R.C.A. and Wu, R. (1983) Nud. Acids Res. 11(16):5521-40. 5. Henikoff, S. (1984)
Gene 28:351-359. 6. Golomb, M. and Chamberlin M.J. (1977) J.of Virol 21(2):743-752. 7. Bailey, J.N., Klement, J.F., McAllister,
W.T., Proc. Natl. Acad. Sci. (1983) 80:2814-2818.
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(gIRL Press