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mp18/19, etc., all of which have a number of highly useful features. All contain a

synthetic MCS, which is located in thelacZgene without disruption of the reading frame

of the gene. This allows efficient selection to be undertaken based on the technique of

blue/white screening (Section 6.3.1). As the series of vectors were developed the number

of restriction sites was increased in an asymmetric fashion. Thus M13mp8, mp12, mp18

and sister vectors which have the same MCS but in reverse orientation, M13mp9, mp13

and mp19 respectively have more restriction sites in the MCS making the vector more

useful since greater choice of restriction enzymes is available (Fig. 6.22). However, one

problem frequently encountered with M13 is the instability and spontaneous loss of

inserts that are greater than 6 kb.

Phagemidsare very similar to M13 and replicate in a similar fashion. One of the first

phagemid vectors, pEMBL, was constructed by inserting a fragment of another phage

termed f1 containing a phage origin of replication and elements for its morphogenesis

into a pUC8 plasmid. Following superinfection with helper phage the f1 origin is

activated allowing single-stranded DNA to be produced. The phage is assembled into a

phage coat extruded through the periplasm and secreted into the culture medium in a

similar way to M13. Without superinfection the phagemid replicates as a pUC type

plasmid and in the replicative form (RF) the DNA isolated is double-stranded. This allows

further manipulations such as restriction digestion, ligation and mapping analysis to be

performed. The pBluescript SK vector is also a phagemid and can be used in its own right

as a cloning vector and manipulated as if it were a plasmid. It may, like M13, be used in

nucleotide sequencing and site-directed mutagenesis, and it is also possible to produce

RNA transcriptsthat may beusedin the productionof labelled cRNA probesorriboprobes

(Section 6.4.2).

6.3.4 Cosmid based vectors

The way in which the phagelDNA is replicated is of particular interest in the

development of larger insert cloning vectors termed cosmids (Fig. 6.23). These are

HindIII

PstI

HincII

AccI

SalI

BamHI

XmaI

SmaI

EcoRI

HindIII

PstI

HincII

AccI

SalI

XbaI

BamHI

XmaI

SmaI

SstI

EcoRI

EcoRI

SmaI

XmaI

BamHI

SalI

AccI

HincII

PstI

HindIII

EcoRI

SstI

SmaI

XmaI

BamHI

XbaI

SalI

AccI

HincII

PstI

HindIII

HindIII

SphI

PstI

HincII

AccI

SalI

XbaI

BamHI

XmaI

SmaI

KpnI

SstI

EcoRI

EcoRI

SstI

KpnI

SmaI

XmaI

BamHI

XbaI

SalI

AccI

HincII

PstI

SphI

HindIII

M13 Multiple Cloning Site/Polylinker

mp8 mp9

mp12 mp13

mp18 mp19

Fig. 6.22Design and orientation of polylinkers in M13 series. Only the main restriction enzymes are indicated.

218 Recombinant DNA and genetic analysis