DNA Cloning

What is DNA Cloning?

DNA cloning can be described as the process of making multiple, identical copies of a particular piece of the genetic material or DNA fragment. In a typical DNA cloning process, the gene or other the target DNA fragment is first inserted into a circular piece of DNA known as a plasmid. This is done using restriction enzymes that “cut and paste” the DNA. It produces a molecule of recombinant DNA.

Next, the recombinant plasmid is introduced into a bacterial cell. The bacteria carrying the plasmid are selected and grown-up. As they reproduce, they replicate the plasmid and pass it onto their offspring, making copies of the rDNA or recombinant DNA it contains.

In some cases, we need several DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used to produce Detection of recombinant clones: From the large number of colonies produced the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.

Gene Cloning Methods

There are Four Major Gene Cloning Techniques, These are Summarised Below:

1. Isolation of DNA to be Cloned: The target DNA may be genomic DNA or complementary DNA or synthetic. The genomic DNA of interest, if contained in a particular restriction fragment that can be isolated from the gel after electrophoresis. Otherwise, a complementary DNA or cDNA fragment is prepared directly by using an mRNA template. The polyadenylated mRNAs are separated from other types of RNAs through affinity column chromatography.

These mRNAs are then copied to cDNAs with the help of reverse transcriptase. As the cDNA is obtained from mRNA in this case, it must contain the uninterrupted coding sequence of a gene and the rDNA molecule will synthesise the eukaryotic gene product in a prokaryotic cell. 

2. Insertion of a Foreign DNA Fragment into a Vector: The isolated cDNA is fragmented by using a specific restriction enzyme to develop specific cohesive ends. The cloning vector is also treated with the same restriction enzyme, so the cohesive ends that are generated are similar to the cDNA

For insertion of a ds-cDNA (double-stranded cDNA) into a cloning vector, it is necessary to add to both termini single-stranded or ss-DNA sequence which should be complementary to a tract of DNA at the termini of a linearised vector. To get the efficient formation of rDNA molecules, the addition of sticky ends on both termini is necessary.

3. Transfer of rDNA into a Bacterial Cell: Before the rDNA can be multiplied by cloning, it must be taken up by a suitable bacterial host cell, which is then transformed, i.e., a host bacterial cell must accept the plasmid with the foreign gen and start transcribing that gene.

The event of entering the plasmid with foreign DNA into the cell is known as “transformation”. A mild heat shock is given to the mixture resulting in the uptake at a higher frequency of the DNA. The selection of transformed cells is carried out by allowing the bacteria to grow in the antibiotic selection medium.

4. Detection of Recombinant Clone: The next step is to select or screen out the few colonies which contain the recombinant plasmid - the use of antibiotics is one of the easiest and useful methods for this purpose. The transformed bacterial cells are plated on a selection medium containing different antibiotics.

The colonies which grow can be said to have a plasmid, as the antibiotic resistance gene of plasmid enables the bacteria to grow. For example, the plasmid pBR 322 contains genes for ampicillin resistance (ampr) and tetracycline resistance (tetr). Thus the trans-formants can be detected by their plating potential on medium containing either (or both) of these antibiotics.

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The Cloning Process

DNA cloning is used in several applications. For example, let's see how DNA cloning is utilised to synthesise a protein (such as human insulin) in bacteria. The basic gene cloning steps are:

1. Cutting and Pasting DNA: A restriction enzyme that recognises a specific target sequence of DNA cuts it into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair with each other. In the case that they won't combine to form an unbroken DNA molecule, they are joined by DNA ligase which is also known as the molecular glue. DNA ligase seals gaps in the DNA backbone.

Using a carefully chosen restriction enzyme, the DNA is digested which gives a:

• The plasmid with a single cut site

• The target gene fragment with a cut site near each end

Then, the fragments are combined with DNA ligase, which links them to make a recombinant plasmid containing the gene.

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2. Bacterial Transformation and Selection: Plasmids and other DNA fragments can be introduced into bacteria, such as the E. coli used, in a process called transformation. 

During this process, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.

A plasmid generally contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Hence, bacteria that take up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid gene will die, while bacteria carrying the plasmid gene can live and reproduce. 

Not all colonies contain the right plasmid. That’s because, during ligation, the DNA fragments do not always get pasted in exactly the way as intended. So, several colonies are checked. Methods like restriction enzyme digestion and Polymerase Chain Reactions are commonly used to check the plasmids.

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3. Protein Production: Once a bacterial colony with the right plasmid is screened, it can be grown to a large culture of plasmid-bearing bacteria.

The bacteria serve as factories producing large amounts of protein. For instance, if the plasmid contains the human insulin gene, the bacteria would start transcribing the gene and translating the mRNA to produce many molecules of human insulin protein.

Once the protein has been produced, the bacterial cells can be lysed to release it. Many other proteins and macromolecules are floating around in bacteria besides the target protein (e.g., insulin). Due to this, the target protein needs to be purified or separated from the other contents of the cells by various biochemical techniques. 

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Application of Gene Cloning

There are many uses of gene cloning in the medical and research industry. Some of the examples include:

• Biopharmaceuticals: DNA cloning is used to produce human proteins with biomedical applications. One example is the production of insulin using E. coli. Other examples include human growth hormones to treat patients who cannot naturally produce this hormone and tissue plasminogen activator which is used to treat strokes.

• Gene Therapy: Gene therapy is used to provide a specific gene that is not available in a patient’s body due to a genetic disorder. DNA cloning was used to build plasmids containing a normal version of the gene that's non-functional in cystic fibrosis. After the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deterioration rate was decreased.

• Gene Analysis: Biologists often use DNA cloning to create artificial recombinant versions of genes that help them understand the function of that specific gene and how and it functions in an organism.