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Chp 1
Basic principles of Gene Cloning
What is cloning?
Molecular cloning
Cell cloning
Organism cloning
What is gene cloning?
What is gene cloning?
What is PCR?
http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html
Kary Mullis invented the polymerase chain reaction (PCR)
during a drive along the coast of California one evening in 1985.
1993 Nobel Prize in Chemistry
PCR reaction mixture
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Primers (18-25 bp)
dNTPs
MgCl2 (cofactor for the enzyme)
Polymerase enzyme
Buffer
Distilled water
So, gene isolation by clonning techniques is still in use.
Chapter 2
Vectors for Gene Clonning: Plasmids
and Bacteriophages
Vectors need to have two important characters :
1- It must be able to replicate within the host cell
2- It must be relatively small, less than 10 kb.
Two kinds of DNA molecule that satisfy these
criteria:
Plasmids and Bacteriophages
• Plasmids almost always carry one or more genes,
and often these genes are responsible for a useful
characteristic that gains to the bacteria
advantage.
• For example, the ability to survive in normally
toxic concentrations of antibiotics such as
chloramphenicol or ampicillin is often due to the
presence in the bacterium of a plasmid carrying
antibiotic resistance genes.
• This is used as a selectable marker in culture.
Conjugative and non-conjugative
Plasmids
Compatibility
• Several different kinds of plasmid may be found in a
single cell, including more than one different
conjugative plasmid at any one time
• In fact, cells of E. coli have been known to contain up to
seven different plasmids at once
• To be able to coexist in the same cell, different
plasmids must be compatible
• If two plasmids are incompatible then one or the other
will be rapidly lost from the cell
• Different types of plasmid can there- fore be assigned
to different incompatibility groups on the basis of
whether or not they can coexist
• Plasmids fall into five different categories:
– 1. Fertility plasmids-allow bacteria to mate to each other
– 2. Resistance plasmids-confer resistance to antibiotics
or toxins
– 3. Degradative plasmids-enable the digestion of unusual
substances
– 4. Col-plasmids-encode colicines which are proteins
that kill other bacteria
– 5. Virulence plasmids-turn a bacterium into a
pathogenic strain
Several eukaryotic viruses have been employed as cloning
vectors for specialized applications: for example,
• human adenoviruses are used in gene therapy (p. 259),
• baculoviruses are used to synthesize important
pharmaceutical proteins in insect cells (p. 240),
• caulimoviruses and geminiviruses have been used for
cloning in plants (p. 120).
These vectors are discussed more fully in Chapter 7.