A new plasmid vector, pSCANS, has been constructed which allows rapid
generation of an ordered set of nested deletions from either strand of a
cloned DNA fragment.
pSCANS is based on the low-copy F replicon.
The size of the vector DNA has been reduced to the 4.4-kbp range by removing
the 2.5-kbp sop (stability of plasmid genes) region.
The resulting plasmid has the low copy number typical of F plasmids and it
remains stable enough to be easily maintained by growth in the presence of
kanamycin, the selective antibiotic.
DNA in amounts convenient for sequencing is readily obtained by amplification
from an IPTG-inducible P1 lytic replicon.
multiple cloning region (MCR),
which has several unique sites
for both shotgun and directional cloning, is flanked on one side by
recognition sequences for the extremely rare cutting intron encoded
nucleases I-CeuI and I-SceI, and on the other side by a recognition
sequence for another intron encoded enzyme, PI-PspI and a nicking site
for the phage f1 protein, gpII, that initiates f1 rolling circle DNA
Cleavage with the intron encoded enzymes leaves four-base 3' overhangs that
are resistant to digestion with E. coli ExoIII.
Between these sites and the MCR are recognition sites for several rare 8-base
cutters that leave ExoIII sensitive termini.
Double cutting with one intron encoded enzyme and an
adjacent rare cutting restriction endonuclease allows for unidirectional
3' to 5' digestion across the insert with ExoIII.
Alternatively, plasmid linearized with I-SceI can be blunt ended to produce
an ExoIII sensitive end and then cut with I-CeuI to generate an ExoIII
resistant end on one side of an insert.
The f1 nicking site can be used for ExoIII digestion of the other strand of
the insert or for producing single-stranded plasmid circles for library
normalization or subtraction.
After ExoIII digestion, the resulting single-stranded regions are digested
with S1 nuclease, and the ends are repaired and ligated with T4 DNA
polymerase and ligase.
Pooling samples from several different ExoIII digestion time points before
subsequent S1 treatment generates a good distribution of deletion clones
Deletion clones are sized and sequenced using vector specific forward and