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flanking sequences. Ideally, the library could be amplified without loss or mis-
representation of sequences, and it could be stored for years without significant
loss of information. Unfortunately, no single vector provides all of these desir-
able attributes.
Partially digested genomic DNA fragments can be cloned into bacterio-
phage λ relatively easily. The disadvantage is that the average λ library of an
insect genome would contain > 100,000 clones, each with an insert that averages
15-20 kb. Cosmid vectors have an advantage in that the size of the inserted DNA
can be two- to three-fold larger; therefore, fewer cosmids need to be evaluated
to find the gene(s) of interest. Thus, cosmids can provide a significant advantage
when it is important to work with an entire gene and its flanking sequences.
Genomic libraries constructed from random fragments of DNA are called
shotgun libraries . To generate a genomic library for an insect may require 10 6
clones to ensure that all sequences in the genome are represented. The library
only can be read if there is a key to open it. The key to libraries is some sort of
probe (see Section 6.6).
6.3.1 Lambda ( λ ) Phage as a Vector
Lambda ( λ ) is a genetically complex, but well-studied, double-stranded-DNA bac-
teriophage of E. coli . The entire DNA sequence of the λ chromosome has been
determined. Nearly 40% of the 48.5-kb chromosome is not essential for propa-
gating the phage in its host. If this nonessential DNA is removed, approximately
the same amount of exogenous DNA can be inserted. At each end of the linear
DNA molecule there are short, single-stranded 5 -projections of 12 nucleotides,
called cos sites , that are complementary to each other in sequence.
The cos sites enable the λ chromosome to circularize after the linear phage is
injected into its E. coli host ( Figure 6.2 ). After replication within the host cell, the
λ DNA is in a linear form when it is packaged into a protein coat. The protein coat
consists of an icosahedral head and a tail that ends in a tail fiber. The infective
phage thus consists of the DNA molecule plus a protein head and tail. The protein
coat allows phage particles to ad sorb by the tip of their tail fiber to receptor sites
on the outer membrane of their E. coli host cells. Adsorption is temperature inde-
pendent, and is dependent upon the presence of magnesium ions.
A wild-type λ has two phases to its life cycle: temperate and lytic. Although
the temperate or lysogenic phase is of little interest to the genetic engineer, λ
which has been genetically modified to serve as a vector retains many character-
istics of the second, or lytic, phase.
 
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