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Figure 6.3 Schematic outline of a bacteriophage λ replacement vector. A linear molecule contains
the cos sites, a left (L) and right (R) arm, and a “stuffer” region with nonessential DNA. The vector is
digested with an appropriate restriction enzyme, the stuffer fragment is removed, and the two arms
anneal. Exogenous DNA that has been cleaved with an appropriate restriction enzyme is added, and
the fragment is ligated in. Exogenous DNA fragments of 18 to 22 kb can be incorporated because
these molecules can be successfully packaged by in vitro packaging. Escherichia coli is infected with the
λ , and thousands of individual plaques are produced. Each plaque contains many thousands of rep-
licas (clones) of a single phage containing exogenous DNA.
and replacement. Insertion vectors have a single target site at which foreign
DNA can be inserted, whereas replacement vectors have a pair of sites defining
a fragment that can be removed and replaced by foreign DNA ( Figure 6.3 ).
Once exogenous DNA has been inserted into the λ vector, this molecule can
be multiplied (cloned) by inserting it into host E. coli cells in one of two ways:
transfection and in vitro packaging. Naked λ DNA (lacking a protein coat) can
be introduced into E. coli cells in a process called transfection. Transfection is
the infection of bacteria by viral nucleic acid alone. The efficiency of transfec-
tion is > 10 4 recombinant clones per microgram of donor DNA. This efficiency
would suffice for the construction of genomic libraries from species with small
genomes. However, larger genomes, such as those of insects, require a more
efficient method of inserting the vector DNA into E. coli . The way to increase
efficiency in introducing recombinant λ DNA molecules into E. coli is called in
vitro packaging . By incorporating the recombinant DNA molecules into phage
protein coats, E. coli can be infected much more readily, thereby increasing the
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