Gene Cloning

Gene Identification and DNA Libraries 97
4.12 Cloning with Bacteriophage λ
If you want to use a λ replacement vector to make a DNA library, you firstly
need to cut out the stuffer fragment. Replacement vectors are usually
designed with multiple cloning sites at the margins of the stuffer fragment to
make this easy (Figure 4.6b). Digesting with a second restriction enzyme,
which only cuts the stuffer fragment, will reduce the stuffer fragment to a
number of small fragments. The sticky ends of these smaller fragments are
not compatible with those of the λ cloning arms so the stuffer fragment is
unlikely to be involved in the recombinants. Fragments of genomic DNA in
the region of up to 23 kb with compatible sticky ends are ligated with the λ
arms to form concatemers which can then be packaged in vivo into bacteriophage
particles and used to transfect E. coli. You will see from Figure 4.7
that the concatemeric DNA which is produced in the ligation reaction can
contain λ arms joined together without insert molecules in place of the
stuffer fragment. These non-recombinant molecules are not wanted in the
DNA library, since they increase the number of plaques that need to be
screened to identify the plaque containing the clone of interest. However, as
the cos sites in these molecules will be less than 37 kb apart they will not be
packaged successfully into phage heads and will not be present in the library.
Many λ vectors use this type of size selection for recombinant molecules.
λ replacement vector
B
B
Digest
B
B
Stuffer
fragment
Digest
λ arms
Insert
Ligate
Concatameric
DNA
λ arms ligated
without insert
λ arms ligated
with insert
Figure 4.7 Cloning with a bacteriophage λ replacement vector. Linear
bacteriophage λ is cut with a restriction enzyme to separate the λ arms and a
stuffer fragment. Digestion with a second restriction enzyme which only cuts the
stuffer fragment reduces the chances of it religating to the arms in the ligation
step. The λ arms are mixed with insert DNA and ligated. Concatemers are
formed consisting of λ arms and insert DNA. Because there is a minimum
distance between cos sites for efficient packaging of λ DNA any arms which
ligate to each other will not package successfully.