There are three different methods by which Recombinant DNA
is made.
1. Transformation
The first step in transformation is to select a piece
of DNA to be inserted into a vector. The second step is to cut that piece of
DNA with a restriction enzyme and then ligate the DNA insert into the vector
with DNA Ligase. The insert contains a selectable marker which allows for
identification of recombinant molecules. An antibiotic marker is often used so
a host cell without a vector dies when exposed to a certain antibiotic, and the
host with the vector will live because it is resistant.
The vector is inserted into a host cell, in a process
called transformation. One example of a possible host cell is E. Coli. The host
cells must be specially prepared to take up the foreign DNA.
Selectable markers can be for antibiotic resistance,
color changes, or any other characteristic which can distinguish transformed
hosts from untransformed hosts.
2. Non-Bacterial
Transformation
This is a process very similar to Transformation, which was described
above. The only difference between the two is non-bacterial does not use
bacteria such as E. Coli for the host.
In microinjection, the DNA is injected directly into the nucleus of the
cell being transformed. In biolistics, the host cells are bombarded with high
velocity microprojectiles, such as particles of gold or tungsten that have been
coated with DNA.
3. Phage Introduction
Phage introduction is the process of transfection,
which is equivalent to transformation, except a phage is used instead of
bacteria. In vitro packagings of a vector is used. This uses lambda or MI3
phages to produce phage plaques which contain recombinants. The recombinants
that are created can be identified by differences in the recombinants and
non-recombinants using various selection methods.
