Biochemical Techniques and Instrumentation

6.2.3.1. Molecular cloning

Molecular cloning or DNA cloning is a process of creating multiple copies of a defined DNA sequence. It amplifies the DNA fragments containing whole genes as well as any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of applications starting from genetic fingerprinting to large scale protein production.

Molecular cloning is also known as recombinant DNA technology. In this process, a piece of DNA or a gene from one organism is transferred to a self replicating genetic element such as a bacterial plasmid, which is a cloning vector. The cloning vector is a small piece of DNA into which a foreign DNA fragments can be inserted. The plasmid with inserted DNA sequence is then transferred to a bacterium, where multiple copies of the same piece of DNA or gene are generated. The sequence must be linked to the origin of replication of the bacterial plasmid for amplification. The origin of replication is a sequence of DNA capable of directing the propagation any linked sequence.

Cloning of DNA fragment involves four steps:

1. Fragmentation

Fragemenation means breaking apart a strand of DNA. The DNA fragment of suitable size has to be isolated, amplified into their product and cut into smaller fragments using restriction enzymes.

2. Ligation

Ligation means gluing together pieces of DNA in a desired sequence. In this step, the DNA fragment is inserted into a plasmid vector. The vector must be first linearised using same restriction enzymes used to cut the DNA and incubated with the DNA fragment under appropriate conditions with an enzyme called DNA ligase.

3. Transfection

Transfection means inserting the newly formed pieces of DNA into cells. A number of techniques are available for transfection and they include chemical sensitivation of cells, electroporation, optical injection and biolistics.

4. Screening/selection

The transfected cells are then cultured in the desired medium for selecting the cells that are successfully transfected with the vector construct containing the desired DNA sequence. Modern cloning vectors contain selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected to grow. In addition, the cloning vectors also contain colour selection markers, which provide blue/white screening on X-gal medium. Recombinant plasmid will appear white or colourless in the presence of X-gal, whereas an intact non-recombinant plasmid will be blue since its gene is fully functional and not disrupted. Further investigation can be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.