Cosmids, phasmids and other advanced vectors - Principles of Gene Manipulation - page 67

Ap R

cos

c2XB
(6.8 kb)

Km R

Sma I

cos

rep

Bam H I

Target genomic
DNA

Sma Bam Bam Sma

(Blunt) (Blunt)

Ap R rep

cos cos

(1) Partial Sau 3A digestion

(2) Phosphatase

Sau 3A Sau 3A

Mix
L igate

32-45 kb

Fig. 5.3 Cosmid cloning scheme of
Bates and Swift (1983). The cosmid c2XB
contains two cos sites, separated by a site
for the restriction endonuclease Sma I which
creates blunt ends. These blunt ends ligate
only very inefficiently under the conditions
used and effectively prevent the formation
of recombinants containing multiple
copies of the vector.

(B) (B)

Sma
(Blunt)

Sma
(Blunt)

Ap R rep

cos cos

37-52 kb

Only packageable molecules

Package
in vitro

of clones containing short foreign DNA or multiple
vector sequences.
An alternative solution to these problems has
been devised by Bates and Swift (1983) who have
constructed cosmid c2XB. This cosmid carries a
Bam HI insertion site and two cos sites separated by a
blunt-end restriction site (Fig. 5.3). The creation of
these blunt ends, which ligate only very inefficiently
under the conditions used, effectively prevents
vector self-ligation in the ligation reaction.
Modern cosmids of the pWE and sCos series (Wahl
et al. 1987, Evans et al. 1989) contain features such
as: (i) multiple cloning sites (Bates & Swift 1983,
Pirrotta et al. 1983, Breter et al. 1987) for simple
cloning using non-size-selected DNA; (ii) phage

promoters flanking the cloning site; and (iii) unique
Not I, Sac II or Sfi l sites (rare cutters, see Chapter 6)
flanking the cloning site to permit removal of the
insert from the vector as single fragments. Mam-
malian expression modules encoding dominant
selectable markers (Chapter 10) may also be present,
for gene transfer to mammalian cells if required.

BACs and PACs as alternatives to cosmids

Phage P1 is a temperate bacteriophage which has
been extensively used for genetic analysis of
Escherichia coli because it can mediate generalized
transduction. Sternberg and co-workers have devel-
oped a P1 vector system which has a capacity for