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DNA cloning is a technique to reproduce DNA fragments. It can be
achieved by two different approaches: (1) cell based, and
(2) using polymerase chain reaction (PCR).
In the cell-based approach, a vector
is required to carry the DNA fragment of interest into the host cell. The
following figure shows a typical procedure by using plasmids
as the cloning vector.
Figure 9-A-1. The essential steps in DNA
cloning using plasmids as vectors.
(a) DNA recombination. The DNA fragment to be cloned is inserted into a
vector (more information). The recombinant
vector must also contain an antibiotic-resistance
gene (not shown).
(b) Transformation. The recombinant DNA enters into the host cell and
proliferates. It is called "transformation" because the function
of the host cell may be altered. Normal E. coli cells are difficult to take up plasmid DNA from the
medium. If they are treated with CaCl2, the transformation
efficiency can be significantly enhanced. Even so, only one cell in
about 10,000 cells may take up a plasmid DNA molecule.
(c) Selective amplification. A specific antibiotic is added to kill E.
coli without any protection. The transformed E. coli is
protected by the antibiotic-resistance
gene whose product can inactivate the specific antibiotic. In this figure,
the numbers of vectors in each E. coli cell are not the same, because
they may also reproduce independently.
(d) Isolation of desired DNA clones.
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