Chp 1Basic principles of Gene Cloning

What is cloning?

Molecular cloningCell cloningOrganism 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

• 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.