Elementary plant Biochemistry and Biotechnology (2+1)

Vectors

• A DNA molecule needs to display several features to be able to act as a vector for gene cloning. Most importantly it must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells. A cloning vector also needs to be relatively small, ideally less than 10 kb in size, as large molecules tend to break down during purification, and are also more difficult to manipulate. Two kinds of DNA molecule that satisfy these criteria can be found in bacterial cells: plasmids and bacteriophage chromosomes.

Plasmids

• Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell. Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium. 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. In the laboratory, antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid.



DNA manipulative enzymes can be grouped into four broad classes, depending on the type of reaction that they catalyze:

1. Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules.
2. Ligases join nucleic acid molecules together.
3. Polymerases make copies of molecules.
4. Modifying enzymes remove or add chemical groups.
Vector types and insert that can be inserted into the vector

Vector type	Insert size
Plasmid	< 5 Kb (but upto 20 Kbp has been tried)
Phagemid	< 5 Kb (but upto 20 Kbp has been tried)
Bacteriophage λ-insertion vector	0-10 Kbp
Bacteriophage λ- replacement vector	9-23 Kbp
Cosmid	30-45 kbp
Bacterial artificial chromosome - BAC	> 300 Kbp
Bacteriophage P1 derived artificial chromosome -PAC	~ 100 Kbp
Yeast artificial chromosome - YAC	> 2000 Kbp