An overview of RDT and the steps involved
Recombinant DNA Technology has the same process as DNA Cloning and Molecular cloning as well as gene cloning. This process involves transferring DNA fragments that are of interest to a scientist from one organism to another self replicating and genetic element.
In addition to this the DNA can be propagated within a host cell that is foreign in nature. This is one of the types of cloning that has become very common in molecular biology labs all around the world.
When scientists study genes it is different from cloning humans or animal cloning because the focus is on specific bacteria or cells as opposed to multicellular organisms. Many scientists who use this type of cloning process may only deal with one particular gene as opposed to cloning animals and the focus on the replication of whole organisms and genes.
In order to clone a gene, DNA fragments that house the gene that scientists are interested in become isolated from the chromosomal DNA because of the use of restriction enzymes. After this process is complete the fragment is united with one of the plasmids that is cut with the exact same restriction enzymes. When chromosomal DNA is combined with the cloning vector it is then transformed into recombinant DNA molecules and if these molecules are joined with a suitable host cell then they can be reproduced with the DNA of the host cell.
In terms of the plasmids that are used in this process they can carry a lot of foreign DNA (up to 20,000bp). Other cloning vectors that scientists use in laboratory settings include viruses and bacteria artificial chromosomes as well as artificial yeast chromosomes. In addition to this, The Cosmid is an artificially made cloning vector and this can carry 45kb of DNA whether foreign or not. The Cosmid can also be encased in lambda phage particles in order to infect E coli cells.
Making recombinant DNA
How does recombinant DNA technology work?
The organism under study, which will be used to donate DNA for the analysis, is called the donor organism.
The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments.
The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus).
The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells.
Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone.
A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function.
Cloning allows the amplification and recovery of a specific DNA segment from a large, complex DNA sample such as a genome.
In as much as the donor DNA was cut into many different fragments, most colonies will carry a different recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that the cloning method works because individual recombinant DNA molecules enter individual bacterial host cells, and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated as chemical reagents.
The steps(in brief) involved in rDNA technology are:->(click here for animation)
- Isolation of DNA
- Fragmentation of the DNA using the enzyme Restriction endonucleases
- Isolation of the desired DNA fragment
- Amplification of the gene of interest
- Ligation of the DNA fragment into a suitable vector by the enzyme DNA ligases
- Transfer of DNA into the host cell
- Culturing the host cells on a suitable medium on a large scale
- Extraction of the desired product
- Downstream processing of the products
Image source:
Content Sources:
https://www.bioarts.com/team_carlson.htm
https://www.ncbi.nlm.nih.gov/books/NBK21881/