Recombinant DNA (rDNA) Technology

• Recombinant DNA (rDNA) refers to molecules formed through genetic recombination in laboratory methods such as molecular cloning.
• rDNA involves bringing together genetic material from multiple sources to create sequences not found naturally in the genome.
• rDNA was first achieved in 1973 by Herbert Boyer and Stanley Cohen.
• Boyer and Cohen used E.coli restriction enzymes to insert foreign DNA into plasmids.
• Recombinant DNA technology involves joining together DNA segments from different molecules to create a new recombinant DNA molecule.
• The recombinant DNA molecule can replicate within a cell under certain conditions, either on its own or after integration into a chromosome.
• Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species.
• Recombinant DNA technique is also known as genetic engineering, involving the manipulation of DNA to create new combinations of genetic material.
• Recombinant DNA has applications in medicine, agriculture and industry.

How Recombinant DNA is Made

• Recombinant DNA is made through various methods

Transformation

• The first step involves selecting a piece of DNA to be inserted into a vector.
• The second step involves cutting that piece of DNA with a restriction enzyme and then ligating the DNA insert into the vector with DNA Ligase.
• The insert contains a selectable marker, which allows for identification of recombinant molecules.
• An antibiotic marker is often used, where a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector lives because it is resistant.
• The vector is inserted into a host cell, in a process called transformation.

Non-Bacterial Transformation

• The process is similar to transformation, however the difference is non-bacterial transformation does not use bacteria such as E. Coli for the host.
• In microinjection, the DNA is injected directly into the nucleus of the cell being transformed.
• In biolistics, the host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.

Phage Introduction

• Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria.

Working of Recombinant DNA and protein expression

• Recombinant DNA works when the host cell expresses protein from the recombinant genes.
• A significant amount of recombinant protein may not be produced by the host unless expression factors are added.
• Protein expression depends upon the gene being surrounded by a collection of signals which provide instructions for the transcription and translation of the gene by the cell.
• These signals include the promoter, the ribosome binding site, and the terminator.
• Expression vectors contain foreign DNA

Tools of Recombinant DNA Technology

• Restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome.
• Restriction enzymes are of two types: Endonucleases and Exonucleases.
• Endonucleases cut within the DNA strand whereas exonucleases remove the nucleotides from the ends of the strands.
• Polymerases help to synthesize, and ligases help to bind.
• Vectors help in carrying and integrating the desired gene, and carry the desired gene into the host organism.
• Plasmids and bacteriophages are the most common vectors in recombinant DNA technology, and have a very high copy number.
• Vectors consist of Origin of Replication (ORI), Selectable Marker and Cloning Sites.
• The host is engineered with the desired DNA with the help of the enzymes, that has recombinant DNA introduced into it.

Steps in Recombinant DNA Technology

• Selection and isolation of DNA insert takes place.
• Selection of suitable cloning vector.
• Introduction of DNA-insert into vector to form rDNA molecule is performed
• rDNA molecule is introduced into a suitable host
• Selection of transformed host cells follows introduction of rDNA molecule
• Last step: expression and multiplication of DNA-insert in the host.

Application of rDNA

• The applications of recombinant DNA are:
• Production of transgenic plants and animals.
• Production of hormones and vaccines.
• Biosynthesis of interferon.
• Antibiotics production.
• Enzyme and Genetic Engineering.
• Prevention and diagnosis of diseases.
• Gene therapy and forensic science
• Environment protection.