Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

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Figure 5.13
Strategies for the selection of recombinant phage.
Part I The Basic Principles of Gene Cloning and DNA Analysis
(a) Insertional activation of the lacZ’ gene
(b) Insertional activation of the λ cl gene
(c) Selection using the Spi phenotype
P2 prophage
(d) Selection on the basis of λ
genome size
Correct size
for packaging
Agar + X-gal + IPTG
Clear plaque = recombinant
Blue plaque = non-recombinant
Clear plaque = recombinant
Turbid plaque = non-recombinant
Too small
too package
Only recombinant λ
phage can infect
Non-recombinant λ
– cannot infect
cos sites
λ catenane
inhibition). Some e cloning vectors are designed so that insertion of new DNA causes
a change from Spi + to Spi − , enabling the recombinants to infect cells that carry P2
prophages. Such cells are used as the host for cloning experiments with these vectors;
only recombinants are Spi − so only recombinants form plaques (Figure 5.13c).
5.4.4 Selection on the basis of * genome size
The e packaging system, which assembles the mature phage particles, can only insert
DNA molecules of between 37 and 52 kb into the head structure. Anything less than
37 kb is not packaged. Many e vectors have been constructed by deleting large segments
of the e DNA molecule (p. 98) and so are less than 37 kb in length. These can only be
packaged into mature phage particles after extra DNA has been inserted, bringing the
total genome size up to 37 kb or more (Figure 5.13d). Therefore, with these vectors only
recombinant phages are able to replicate.