Biology Reference
In-Depth Information
compared with the desired fragment size, a
partial
digestion will produce a set
of overlapping fragments. Ideally, these fragments will be a nearly random array
of the entire genome. However, it is possible that some regions of the genome
will not be represented because they lack the appropriate cleavage sites for the
enzyme used, or the DNA may not be cleaved with equal efficiency, particularly
in heterochromatic regions containing repetitive elements. It is especially difficult
to clone telomeres and centromeres, for example. Furthermore, some regions of
the genome may be toxic to their
E. coli
host, so no clones will be produced.
After the genomic DNA has been partially digested with an appropriate
restriction endonuclease, the DNA fragments are size-fractionated by centrifu-
gation or by gel electrophoresis. This separates out the DNA fragments greater
than or smaller than 18-22 kb. Preparing a representative library requires high-
quality vector DNA. Large-scale preparation of
λ
DNA should yield pure prepara-
tions that lack the central stuffer region, or it can reinsert back into the vector
later on. Removal of the central stuffer region is carried out by centrifugation,
elution, or electrophoretic separation. However, it is difficult to remove all of
the stuffer fragments, so it is important to determine, by appropriate controls,
how often the stuffer is reinserted back into the vector.
Optimizing concentrations of vector and exogenous will result in adequate liga-
tions and thus a good library. Because a portion of the DNA molecules to be ligated
will have damaged ends, the ratio of vector to insert DNA will probably have to be
determined empirically in small reactions. It is desirable to produce long concate-
nated molecules that can be cut at the
cos
sites and packaged. Likewise, the appro-
priate ratios of ingredients used for
in vitro
packaging will have to be determined
empirically. Once the DNA has been packaged, the phage can be stored at 4°C for
years. Alternatively, the phage can be amplified by multiplication in
E. coli
.
Commonly used vectors derived from
λ
include the gt and EMBL series.
λ
gt10
was designed for cloning short DNA fragments, especially cDNA.
λ
gt11 is used
to construct cDNA libraries, as described in Section 6.4. DNA properly aligned
with the
lacZ
gene in
λ
gt11 will be expressed in
E. coli
as a
fusion protein
. EMBL
vectors are a family of replacement vectors that provide a high level of repro-
duction in
E. coli
, polylinker cloning sites, and the ability to select for recom-
binant phage. EMBL3 and EMBL4 vectors, or their derivatives, are particularly
useful for constructing genomic libraries.
6.3.2 Cloning with Cosmids
Cosmids are engineered vectors that combine characteristics of both plasmids
and phage. They have been constructed to include a fragment of
λ
that includes
