Tools of Recombinant DNA Technology

Recombinant DNA research is based on the isolation and manipulation of DNA, including end-to-end joining of sequences from different sources to produce chimeric DNA.

Progress in recombinant DNA technology became possible after the discovery of restriction enzymes and the development of methods to produce large quantities of gene-containing DNA fragments using plasmid, phage, and other vectors.

Main tools:

The key tools involved are enzymes, cloning vectors, and competent host organisms.

Recombinant DNA work depends on the ability to cut DNA molecules at defined sequences.

• Enzymes are required both to cut the desired gene and to cleave the vector.
• DNA fragments are then brought together to form covalently linked chimeric molecules through ligation catalysed by DNA ligase.

Restriction Enzymes

• Act as molecular scissors; cut DNA at specific sites into identifiable fragments.
• Originally found in bacteria as a defence against bacteriophages.

Belong to the class nucleases, which include:

• Exonucleases - remove nucleotides from the ends of DNA.
• Endonucleases - make cuts at specific positions within DNA.

DNA Ligase

• Joins/seals DNA fragments to form recombinant DNA.
• Isolated from E. coli (75 kDa) and T4 phage (68 kDa).

Other Enzymes

• Alkaline phosphatase - removes 5′ phosphate group; prevents self-ligation of vector.
• DNA polymerase - synthesises complementary DNA (cDNA) in vitro.

A DNA molecule that carries foreign DNA into a host cell and replicates independently. Also called vector DNA, transport DNA, or carrier DNA.

Types: Plasmids, Bacteriophages, Cosmids, Phasmids.

Essential features:

• Ori - origin of replication.
• Selectable markers - identify transformants; eliminate non-transformants.
• Cloning sites - restriction sites for inserting foreign DNA; insertion here causes insertional inactivation.

Vectors by organism:

A competent host cell is a cell that can take up foreign DNA from its surroundings.

• DNA is hydrophilic and cannot pass through membranes naturally.
• Cells must be made competent (able to take up foreign DNA).
• Methods: Heat shock, Microinjection, Biolistics (gene gun).

• Recombinant DNA technology manipulates and joins DNA from different sources using three main tools: enzymes, cloning vectors, and competent hosts.
• Restriction enzymes act as "molecular scissors" to cut DNA at specific sites, while DNA ligase acts as "glue" to seal the fragments together.
• Cloning vectors (such as plasmids or bacteriophages) serve as vehicles for carrying, inserting, and replicating foreign DNA within a host cell.
• Competent hosts are cells that have been specially treated (using techniques such as heat shock or gene guns) so they can successfully take up the foreign recombinant DNA.