Suppose you have generated important data and you do not want to lose it. What would you do to protect it? You may either file it or store it in a folder. Or you would generate multiple copies of the information using a Xerox. The Xerox machine reads the information in your paper and gives you a printout in the form of a photocopy. Thus, Xerox creates exact replicas of the document. Similarly, our body protects the genetic material by generating multiple copies. An example involves the replication process.

Scientists felt the need to generate multiple DNA copies. The process of cloning involves obtaining multiple copies of a gene or a DNA segment using technology. Each identical copy of a DNA segment or a gene segment is known as a clone. Human genome project utilizes maximum amounts of clones in various experiments. Many such cloned DNA collections make up a clone library. Cloning is not only applicable to DNA or a gene segment but also possible with the entire cell. This type of cloning exploits the natural process of cell division to make many copies of an entire cell. The genetic makeup of the cloned cells is known as cell lines. They are identical to the original cell. Gene cloning involves recombining a population of DNA molecules into the vector. The recombined DNA molecules contain the gene of interest. The DNA is inserted into a vector so that each vector consists of single DNA from the original population. The screening of the colonies revealed the presence or absence of the DNA of interest. Thus, understanding cloning requires knowledge of the clones and their production.





Image 1: Gene Cloning





The review article discusses various aspects of cloning:

1. Restriction enzymes and cloning vectors

2. Collection of clones in the form of a library

3. Screening of specific clones

4. Analyzing cloned DNA through a molecular approach

5. Identifying adjacent clones

6. Applications

Cloning involves producing multiple copies of the genes or DNA segment of interest. In short, the procedure involves digesting a DNA segment with restriction enzymes, introducing it in a vector, transforming the recombinant vector into the host and observing the expression of the gene of interest. The screened clones are later on analyzed using molecular techniques.





Restriction digestion:

The restriction enzymes are used to digest the DNA. These enzymes are endonucleases capable of cleaving specific sites in the DNA known as the restriction sites. The restriction sites have specific nucleotide base pairs. A restriction enzyme cleaves a specific base by breaking the phosphodiester bond. Cleaving of the DNA can be achieved using one or a combination of restriction enzymes. The process of cleaving the DNA to obtain fragments of different sizes is known as restriction digestion. Separation of the DNA fragments is possible with the electrophoretic technique. Smaller fragments travel faster than the larger fragments, thereby forming bands. After visualizing bands, the portion of the gel consisting of a DNA fragment is cut and used for cloning experiments. Here are the two options. One of the options is to make multiple copies of the gene using amplification techniques such as PCR. The second option is to incorporate the DNA into the cloning vector. PCR or polymerase chain reaction is a process of amplifying the gene or a DNA using a sophisticated thermal cycler. Cloning DNA using a vector involves inserting the desired DNA into a plasmid or another vector.





Cloning vectors:

A cloning vector replicates within the host organism. It consists of a restriction site which can be cleaved for inserting the desired DNA fragment. Various cloning vectors are available. A plasmid vector is a plasmid or an extrachromosomal material of the bacteria. It is isolated and used as a vector. It consists of three sites such as the origin of replication, a selectable marker gene, and one or more restriction sites. For example, pUC 19 vector has a polylinker site or multiple cloning sites. On treating the restriction site with a restriction enzyme, that particular site gets cleaved and the desired fragment of DNA gets inserted in that place. Other examples of vectors include shuttle vectors, expression vectors, cosmids, phasmids, and artificial chromosomes. Shuttle vectors are bifunctional. They are capable of insertion into two or more hosts. Expression vectors express the desired gene to get the protein product.





Clone Library:

It can be anything from a collection of clones including cDNAs, chromosomes, and genomes. A cDNA library is nothing but a collection of cloned cDNA sequences synthesized from mRNA. A collection of cloned DNA in which the sequence appears at least once is known as a genomic library. The clone libraries appear analogous to book library. Just as the books provide plenty of information, the clone libraries provide a lot of information about the genome. Genome libraries production involves inserting the required DNA digest into a vector and storing the product. However, there are other methods too. Gene splitting with a restriction enzyme sometimes gives the undesired product. Such cases involve other techniques like mechanical shearing or partial digestion. Large DNA insertion involves mechanical shearing. The process of screening is made easy using chromosome libraries. 24 different human chromosomes libraries are available. The preparation of the cDNA libraries involves mRNA molecules as raw materials. After making clone libraries, a specific gene is found using screening techniques.





Image 2: Transformation of the recombinant plasmid





Screening procedures:

The screening of cDNA libraries involves an antibody probe. The first step is to transform the recombinant plasmid consisting of the required cloned cDNA into a host such as E. coli bacteria. Next step involves plating the bacteria on a selective medium to observe the growth of the colonies. Transferring the colonies into a microtiter well enables the bacteria to grow. The transfer of the colonies to the membrane filter also enables them to grow to get the expression of the gene. Removal of the filter and cell lysis enables the protein product exposure. Radioactively labeled antibody treatment with the protein product is the final step. Autoradiography helps the observation of clones in the form of dark spots. Screening a genomic library involves a similar system. It involves plating the transformed bacteria on a medium and processing them through replica plating. Here, the probed DNA is used instead of a radioactively labeled antibody. Complementation test helps to identify the specific genes.





Analyzing cloned DNA:

Analysis of clones involves molecular biology techniques such as restriction mapping, southern and northern blotting. Restriction mapping includes the physical mapping of the genes. Restriction digestion gives rise to DNA fragments of different sizes. Electrophoresis separates the fragments in the form of bands. Southern blotting uses a nitrocellulose membrane filter placed on the gel immersed in the buffer solution. Capillary action of the blotting paper enables the DNA to come in contact with the buffer and transfer to the membrane filter. The filter gets exposed to probes which hybridize with the DNA. Autoradiography helps to detect the DNA. Southern blotting mainly involves blotting DNA. Northern blotting involves blotting RNA instead of DNA.





Adjacent clone identification:

Genes between the flanking markers are found through chromosome walking. Hence, adjacent clone identification requires chromosome walking. The overlapping clones help to study larger DNA segments.





Applications of cloning:

1. Recombinant DNA technology or genetic engineering: Cloning techniques involve obtaining large amounts of pure DNA. Expression of the desired products including proteins, vitamins, enzymes, growth factors, and other biomolecules is possible with recombinant DNA technology. Cloned genes help to detect mutations. Plant vectors consisting of the desired gene improve the crop quality. Cloning enables development of transgenic plant varieties with stress tolerance, drought tolerance, and herbicide or pesticide resistance. Particular protein synthesis is achieved using expression vectors.

2. Transgenics: These organisms, plants, animals or microbes, involve manipulated genes cloned into several copies. The genes of interest express phenotypically. Examples include knockout animals.

3. Gene therapy: It helps in replacing the gene that has lost its function. The defective gene identification accompanies cloning the normal gene inserted into the affected individual through a vector. The first gene therapy was successful in children suffering from ADA deficiency. The gene therapy is a new way to treat genetic disease because the abnormal gene is corrected or replaced.

4. In vitro fertilization : Microsurgery involves removal of the nucleus from the donor’s oocyte. The microinjection of the surrogate mother’s somatic cell nucleus into the donor’s oocyte makes the cell chimeric. The chimeric cell cultured in the laboratory develops into an embryo. The embryo gets implanted into the surrogate mother’s uterus.





References:

[1] Gene cloning and DNA analysis, T.A. Brown

[2] Recombinant DNA Technology, Sardul Singh Sandhu

[3] Biotechnology-4: Including Recombinant DNA Technology, Environmental, S. Mahesh

[4] Gene Cloning and Manipulation, Christopher Howe

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