Biomedical Engineering Reference
In-Depth Information
P
OH
Ligase
P
OH
+ dimers, etc.
Alkaline
phosphatase
2Pi
OH
OH
Ligase
OHOH
No reaction
HO
P
HO
Ligase
P
Foreign DNA
fragment
Nick
Transformation
Fig. 3.8
Application of alkaline
phosphatase treatment to prevent
recircularization of vector plasmid
without insertion of foreign DNA.
Nick
Host repairs one nick at each
join
one or more restriction endonucleases. One such
molecule is shown in Fig. 3.9. The molecule can be
ligated to both ends of the foreign DNA to be cloned,
and then treated with restriction endonuclease to
produce a sticky-ended fragment, which can be
incorporated into a vector molecule that has been
cut with the same restriction endonuclease. Inser-
tion by means of the linker creates restriction-
enzyme target sites at each end of the foreign DNA
and so enables the foreign DNA to be excised and
recovered after cloning and amplification in the host
bacterium.
the foreign DNA will be cloned as two or more sub-
fragments. One solution to this problem is to choose
another restriction enzyme, but there may not be a
suitable choice if the foreign DNA is large and has
sites for several restriction enzymes. Another solu-
tion is to methylate internal restriction sites with the
appropriate modification methylase. An example of
this is described in Fig. 6.2. Alternatively, a general
solution to the problem is provided by chemically
synthesized adaptor molecules which have a
pre-
formed
cohesive end (Wu
et al.
1978). Consider a
blunt-ended foreign DNA containing an internal
Bam
HI site (Fig. 3.10), which is to be cloned in a
Bam
Hl-cut vector. The
Bam
adaptor molecule has
one blunt end bearing a 5
Adaptors
phosphate group and a
Bam
cohesive end which is not phosphorylated. The
adaptor can be ligated to the foreign DNA ends. The
foreign DNA plus added adaptors is then phosphory-
′
It may be the case that the restriction enzyme used to
generate the cohesive ends in the linker will also cut
the foreign DNA at internal sites. In this situation,
