Biomedical Engineering Reference
In-Depth Information
Short arm
+
pac
loxP
Stuffer
Insert DNA
P1 and plasmid
replicons:
kan
r
, loxP
Long arm
Ligate
P1 and plasmid
replicons:
kan
r
,
loxP
pac
loxP
Insert DNA
Stuffer
115 kb
Pac extract cleaves at
pac
site and inserts DNA into
P1 heads. Tails are attached
Fig. 5.4
The phage P1 vector system.
The P1 vector Ad10 (Sternberg 1990)
is digested to generate short and
long vector arms. These are
dephosphorylated to prevent
self-ligation. Size-selected insert DNA
(85-100 kb) is ligated with vector
arms, ready for a two-stage processing
by packaging extracts. First, the
recombinant DNA is cleaved at the
pac
site by pacase in the packaging extract.
Then the pacase works in concert with
head/tail extract to insert DNA into
phage heads,
pac
site first, cleaving off a
headful of DNA at 115 kb. Heads and
tails then unite. The resulting phage
particle can inject recombinant DNA
into host
E. coli.
The host is
cre
+
. The
cre
recombinase acts on
lox
P sites to
produce a circular plasmid. The plasmid
is maintained at low copy number, but
can be amplified by inducing the P1
lytic operon.
P1 heads
and tails
+
Allow phage to adsorb
to host strain and inject
DNA into
cre
+
host cell
loxP
Cre
recombinase protein circularizes injected DNA at the
loxP
sites. DNA-replicates using
plasmid replicon. Plasmid copy number is increased by induction of P1 lytic replicon.
DNA fragments as large as 100 kb (Sternberg 1990,
Pierce
et al.
1992). Thus the capacity is about twice
that of cosmid clones but less than that of yeast
artificial chromosome (YAC) clones (see p. 159). The
P1 vector contains a packaging site (
pac
) which is
necessary for
in vitro
packaging of recombinant
molecules into phage particles. The vectors contain
two
lox
P sites. These are the sites recognized by the
phage recombinase, the product of the phage
cre
gene, and which lead to circularization of the pack-
aged DNA after it has been injected into an
E. coli
host expressing the recombinase (Fig. 5.4). Clones
are maintained in
E. coli
as low-copy-number plas-
mids by selection for a vector kanamycin-resistance
marker. A high copy number can be induced by
exploitation of the P1 lytic replicon (Sternberg 1990).
This P1 system has been used to construct genomic
libraries of mouse, human, fission yeast and
Droso-
phila
DNA (Hoheisel
et al.
1993, Hartl
et al
. 1994).
Shizuya
et al
. (1992) have developed a bacterial
cloning system for mapping and analysis of complex
genomes. This BAC system (bacterial artificial chro-
mosome) is based on the single-copy sex factor F of
E.
coli
. This vector (Fig. 5.5) includes the
cos
N and P1
lox
P sites, two cloning sites (
Hin
dIII and
Bam
HI) and
several G
λ
C restriction enzyme sites (e.g.
Sfi
I,
Not
I,
etc.) for potential excision of the inserts. The cloning
site is also flanked by T7 and SP6 promoters for
generating RNA probes. This BAC can be trans-
formed into
E. coli
very efficiently, thus avoiding the
+
