Saturday, January 22, 2011

DNA Cloning

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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