The Application of Recombinant DNA Technology

Recombinant DNA technology is one of the pillars of modern biotechnology because it is a method by which a living organism or parts of it are changed or incorporated as a particular characteristic into another living organism. It has become one of the most commercially critical technologies in medicine, agriculture and other industries. We nowadays commonly call this type of technology genetic engineering. It is also a controversial technology and for some a worrying development in the manipulation of the genetic code.

One of the great applications of recmobinant DNA technology is the production of insulin. The others include the quantitative preparation of proteins on a large-scale, production of recombinant vaccines, antenatal diagnosis of genetic disease, monoclonal antibodies, identification of gene mutations, detecting the activation of oncogenes, production of genetically modified crops, bioterrorism detection, cell and tissue culture, xenotransplantation

Genetic recombination is a natural process whereby information is exchanged between two DNA segments. It often occurs within the same species and is one of the reasons for genetic differences between organisms in the same species.

IN 1973, Boyer and Cohen fused two pieces of DNA together and in doing so they created history.  It became recombinant DNA technology when a gene from one species was transferred into another living organism using artificial or synthetic means. 

A piece of recombinant DNA (rDNA) is a unique molecule which is formed by joining two or more pieces of DNA fragments together. These unique molecules would not exist if they had not been constructed in the laboratory. In virtually all cases, the DNA fragments are produced from different species. 

Recombinant DNA then is generated by a process of steps that recombine DNA segments. Under particular conditions, a recombinant DNA molecule is able to enter a cell where it is replicated. This particular attribute makes recombinant DNA technology extremely powerful. The process of reproducing artificially-created DNA is called DNA cloning.

Clones

The term ‘clone’ means a population of identical organisms derived from a single parent. In molecular biology, a collection of molecules or cells which are identical the original molecule or cell is also termed a clone. 

The Process of Creating Recombinant DNA: The Overview

1. A piece of DNA is isolated and purified. The target DNA is usually a gene which is a specific piece of genetic code for a proteins let’s say. This is the target gene. It also includes the vector too into which the target DNA will be inserted. Some target DNA also has to be synthesized in vitro. Restriction endonucleases are used to cut a piece of DNA from the donor DNA which might be a gene or an instruction for a protein. The same type of restriction endonuclease is used to cut the plasmid DNA or some other DNA which is to become the host for the desired piece of DNA.
2. The desired DNA (target gene) is connected into the cut plasmid DNA using a DNA ligase enzyme which seals up the DNA. This is now known as the recombinant DNA.
3. The target gene is inserted into a plasmid or phage. This becomes a plasmid vector or phage vector and is generically called the replicon.
4. The replicon is introduced into a host cell for cloning and either to express a protein or not. The host cell is also known as the recombinant bacterium.
5. The cloned replicon is also referred to as recombinant DNA.
6. The cloning process is needed to produce many copies of the DNA. 
7. The recombinant bacterium multiplies with the gene of interest. These are all clones.  
8. The DNA is expressed to form protein. Large amounts of the protein are produced as a result which would not normally be possible.
9. The protein is purified by whatever downstream process is most suitable.

The Essential Components

Restriction Endonucleases

One of the most important enzymes in creating recombinant DNA technology are the restriction endonucleases. These were discovered by Arber, Smith and Nathans in the early 1970s. Without them there would be no generation of replicons and vectors.

The restriction endonucleases are enzymes found in all bacteria. They protect bacteria from viral infection by breaking up their DNA which destroys them and their ability to replicate. We know of many hundreds. They are named after the bacterium in which they were found. They always take a three-letter abbreviation/ For example, Hae III, Eco R1 and Hin I are classic examples. Eco R1 is an enzyme from Escherichia coli. Hae refers to Haemophilus aegyptius and Hin refers to Haemophilus influenzae.

The reason bacteria do not destroy their own DNA is because this has been modified by methylation of the DNA sequence using a methylase enzyme.

In Eco R1, the strain of E.coli is included in the name where R means the strain of E. coli used. The numerals I,II, III indicate the serial numbers of enzymes from the same bacterium in the order of their discovery.

Each restriction enzyme recognizes a specific base sequence in double-stranded DNA and catalyses the split in the DNA at this site. The base sequence recognised has very special properties: it is 4 to 8 base pairs long and it is palindromic (reads the same from left to right as in MADAM, DAD). EcoR1 for example cuts at the sequence GAATTC and BamH1 cuts at GGATCC.

Type 1 restriction endonucleases

These cut DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognised by the restriction enzyme. These are usually random and imprecise cuts and not very useful for generating recombinant DNA.

Type II

These enzymes cut both strands of DNA within a particular sequence recognised by the restriction endonuclease. They are the most useful for molecular biology. The DNA sequence is symmetrical. It means that they read the DNA in the 5′ to 3′ direction on both strands. The sequence is palindromic.

Some of these enzyme produce ‘blunt ends’ where the cut is in the middle. Others generate ‘sticky ends’ or staggered cuts.

Plasmids

Plasmids are independently replicating circles of DNA. The circles of DNA can only be replicated in bacteria. They are critical because foreign DNA is inserted into the plasmid and then replicated. 

These plasmids used for cloning also carry drug resistance genes that are needed for selection. In reality, they spread antibiotic resistance genes between bacterial species.

One of the first and most versatile plasmid vectors is pBR322 and is the forerunner of plasmid vectors used today in recombinant technology. This plasmid has an origin of replication (ori) and gene (rop) that regulates the number of copies of plasmid DNA in the cell. It has two marker genes that confer resistance to ampicillin and to tetracycline. It also has a number of unique restriction sites that are useful for constructing rDNA.

Bacteriophage Vector 

The phage used as a vector is a derivative of the bacteriophage lambda. They contain linear DNA molecules whose region can be replaced with foreign DNA without disrupting its life cycle. the cloning limit is 8 to 20 kb.

The lambda viral genome is 48.5 kb linear DNA with a 12 base ssDNA ‘sticky end’ at both ends. These ends are complementary in sequence and can be hybridized to each other. This is the cos site meaning cohesive ends.

Infection occurs when the lambda tail fibres adsorb to a cell surface receptor, the cell contracts and the DNA is injected. The DNA circularises at the cos site, and lambda begins its life cycle in the E.coli host.

DNA Ligase

An enzyme used to combine or attach 2 pieces ends of DNA together. It covalently links the two ends of DNA together by forming phosphodiester bonds on the phosphate-sugar backbone.

Particular E.coli Strains For Hosting DNA

Various commercial E .coli strains are available. 

How do we identify the host cells containing the recombinant DNA?

Once the cloning vector which is a plasmid or phase has the gene inserted into it, this recombinant DNA is introduced into the host cell. This may include E. coli.

The transformation of the host cell is not a very efficient way of getting DNA into a cell because only a fraction of these cells ever take up the recombinant DNA. The host cell has to be shocked in a way that causes it to take up the DNA. One way is to heat shock at 42ºC for 45 seconds and immediately add the plasmid.  Those cells that are transformed must be distinguished from the huge majority of untransformed cells. 

identification of these host cells containing the rDNA is known either as genetic selection or screening and they are different processes. In a selection process, the cells are grown under conditions where only the transformed cells survive whilst all the others die. The host cells for example are the only ones which have protection against an antibiotic which is possessed by the vector plasmid.

In a screen, the transformed cells are individually tested for the presence of the desired recombinant DNA. Normally, a number of colonies of cells are first selected and then screened for colonies carrying the desired insert.

Screening Strategies

Four basic techniques are used.

1. Gel electrophoresis – allows separation of vector DNA from cloned fragments.
2. Cloned DNA molecules are sequenced by the dideoxy chain-termination method.
3. The polymerase chain reaction amplifies a specific DNA sequence from a complex mixture.
4. Blotting techniques permit detection of specific DNA fragments and mRNAs with DNA probes.

Types of Blotting techniques

1. Southern Blotting – the first nucleic acid blotting procedure developed in 1975 by Southern. It is used for the specific detection of DNA molecules. 
2. Northern Blotting – a technique for specific identification of RNA molecules.
3. Western blotting – involves the identification of proteins especially in antigen – antibody complexes.