Gene Cloning- Requirements, Principle, Steps, Applications

DNA (deoxyribonucleic acid) is the molecule that carries the genetic information of all living organisms. DNA is composed of four types of nucleotides: adenine (A), thymine (T), cytosine (C) and guanine (G). These nucleotides form a double helix structure, where each strand is complementary to the other. The sequence of nucleotides in a DNA molecule determines the sequence of amino acids in a protein, which in turn determines the structure and function of the protein.

Gene cloning is a technique that allows scientists to produce multiple copies of a specific gene or a segment of DNA. Gene cloning can be used for various purposes, such as studying gene function, producing recombinant proteins, creating transgenic organisms, developing gene therapies, etc. Gene cloning involves the following steps:

• Isolation of the DNA fragment or gene of interest from a source organism
• Insertion of the DNA fragment or gene into a suitable vector, which is a DNA molecule that can replicate independently in a host cell
• Introduction of the vector containing the DNA fragment or gene into a host cell, usually a bacterium or a yeast
• Selection and identification of the host cells that have taken up the vector and contain the DNA fragment or gene
• Multiplication and expression of the DNA fragment or gene in the host cells
• Isolation and purification of the product, which can be either the DNA fragment or gene itself or the protein encoded by it

Gene cloning can be classified into two types: cell-based and cell-free. Cell-based gene cloning involves the use of living cells as hosts for the vectors and the DNA fragments or genes. Cell-free gene cloning involves the use of artificial systems that mimic the cellular environment and allow the synthesis of DNA fragments or genes without using living cells. In this article, we will focus on cell-based gene cloning and its requirements, principle, steps and applications.

Cell-based gene cloning is a technique that involves transferring a gene of interest from one organism to another using a vector and a host cell. The vector is a DNA molecule that can carry the gene of interest and replicate inside the host cell. The host cell is usually a bacterium that can take up the vector and express the gene of interest.

To perform cell-based gene cloning, the following requirements are needed:

• DNA fragment containing the gene of interest: This is the DNA segment that encodes the protein, enzyme, hormone or other product that we want to clone. The gene of interest can be isolated from any source, such as animal, plant, human or microbial cells, using various methods such as restriction digestion, PCR amplification or reverse transcription.
• Restriction enzymes and ligase enzymes: These are enzymes that can cut and join DNA molecules at specific sites. Restriction enzymes recognize and cleave DNA at specific sequences called restriction sites, creating sticky or blunt ends. Ligase enzymes join the sticky or blunt ends of two DNA molecules by forming phosphodiester bonds. These enzymes are used to insert the gene of interest into the vector and to form recombinant DNA molecules.
• Vectors: These are DNA molecules that can carry, maintain and replicate the gene of interest in the host cell. Vectors can be derived from plasmids, bacteriophages, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) or mammalian artificial chromosomes (MACs). Vectors should have some essential characteristics such as self-replication ability, unique restriction sites, marker genes and origin of replication.
• Host cells: These are living cells that can take up the vector and allow its replication and expression. Host cells can be bacteria, yeast, fungi, insect cells, plant cells or animal cells. Host cells should have some desirable properties such as high transformation efficiency, fast growth rate, easy manipulation and low toxicity. The most commonly used host cells for gene cloning are Escherichia coli (E. coli) bacteria.

These are the basic requirements for cell-based gene cloning. By using these components, we can create recombinant DNA molecules that contain the gene of interest and introduce them into host cells for replication and expression. This way, we can obtain multiple copies of the gene of interest or its product for various applications.

Gene cloning is based on the principle of recombinant DNA technology, which involves the manipulation of DNA molecules to create new combinations of genetic material. Recombinant DNA technology allows the transfer of a specific gene or a fragment of DNA from one organism to another, using a vector as a carrier. A vector is a DNA molecule that can replicate independently in a host cell and can carry foreign DNA into it. The most common vectors used for gene cloning are plasmids and bacteriophages.

Plasmids are circular, double-stranded DNA molecules that are found naturally in some bacteria. They can replicate autonomously in the bacterial cell and can be transferred between bacteria by a process called conjugation. Plasmids often carry genes that confer some advantage to the bacteria, such as antibiotic resistance or metabolic capabilities. Plasmids can be modified in the laboratory to insert foreign DNA fragments into them, creating recombinant plasmids. Recombinant plasmids can then be introduced into bacterial cells by a process called transformation, where the bacteria take up the plasmid DNA from the environment. The transformed bacteria can then be selected and grown in large numbers to produce multiple copies of the recombinant plasmid and the foreign DNA.

Bacteriophages are viruses that infect bacteria and use them as hosts to replicate their own genetic material. Bacteriophages have two types of life cycles: lytic and lysogenic. In the lytic cycle, the bacteriophage injects its DNA into the bacterial cell and takes over its machinery to produce new viral particles, which then burst out of the cell and infect other bacteria. In the lysogenic cycle, the bacteriophage integrates its DNA into the bacterial chromosome and becomes dormant, replicating along with the host cell. Bacteriophages can be used as vectors for gene cloning by inserting foreign DNA fragments into their genomes, creating recombinant bacteriophages. Recombinant bacteriophages can then infect bacterial cells and either enter the lytic cycle or the lysogenic cycle, depending on the type of bacteriophage used. The infected bacteria can then be selected and grown in large numbers to produce multiple copies of the recombinant bacteriophage and the foreign DNA.

The principle of gene cloning can be summarized as follows:

1. Isolation of a DNA fragment containing the gene of interest from a donor organism.
2. Insertion of the DNA fragment into a suitable vector to form a recombinant DNA molecule.
3. Introduction of the recombinant DNA molecule into a host cell, usually a bacterium.
4. Selection and multiplication of the host cells that contain the recombinant DNA molecule.
5. Isolation and purification of the cloned gene or its product from the host cells.

Gene cloning has many applications in biotechnology, medicine, agriculture, and research. Some examples of gene cloning are:

• Production of recombinant proteins, such as insulin, growth hormone, vaccines, antibodies, etc.
• Generation of transgenic organisms, such as plants, animals, or microorganisms, that have enhanced traits or novel functions.
• Analysis of gene function and regulation by creating mutants or expressing genes in different contexts.
• Identification and diagnosis of genetic diseases by detecting mutations or variations in genes.
• Discovery and development of new drugs by screening libraries of genes or proteins.

The basic steps involved in gene cloning are:

1. Isolation of the DNA fragment or gene. The target DNA or gene to be cloned must be first isolated. A gene of interest is a fragment of gene whose product (a protein, enzyme or a hormone) interests us. For example, gene encoding for the hormone insulin. The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut DNA at specific recognition nucleotide sequences known as restriction sites towards the inner region (hence endonuclease) producing blunt or sticky ends. Sometimes, reverse transcriptase enzyme may also be used which synthesizes complementary DNA strand of the desired gene using its mRNA.
2. Selection of suitable cloning vector. The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate there along with the GI making its multiple copies. The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the application of the insert the suitable vector is selected. The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes (MACs). However, the most commonly used cloning vectors include plasmids and bacteriophages (phage λ) beside all the other available vectors.
3. Formation of recombinant DNA. The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment. The mixture of donor DNA fragment and plasmid vector are mixed together. In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector occurs. The resulting DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In the terminology of genetics this intermixing of different DNA strands is called recombination. Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the recombinant DNA technology.
4. Transformation of recombinant vector into suitable host. The recombinant vector is transformed into suitable host cell mostly, a bacterial cell. This is done either for one or both of the following reasons: To replicate the recombinant DNA molecule in order to get the multiple copies of the GI; To allow the expression of the GI such that it produces its needed protein product. Some bacteria are naturally transformable; they take up the recombinant vector automatically. For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally competent. Some other bacteria, on the other hand require the incorporation by artificial methods such as Ca++ ion treatment, electroporation, etc.
5. Isolation of recombinant cells. The transformation process generates a mixed population of transformed and non-transformed host cells. The selection process involves filtering the transformed host cells only. For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed. For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene). When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin.
6. Multiplication of selected host cells. Once transformed host cells are separated by the screening process; becomes necessary to provide them optimum parameters to grow and multiply. In this step the transformed host cells are introduced into fresh culture media . At this stage the host cells divide and re-divide along with the replication of the recombinant DNA carried by them. If the aim is obtaining numerous copies of GI, then simply replication of the host cell is allowed. But for obtaining the product of interest, favourable conditions must be provided such that the GI in the vector expresses the product of interest.
7. Isolation and purification of the product. The next step involves isolation of the multiplied GI attached with the vector or of the protein encoded by it. This is followed by purification of the isolated gene copy/protein.

The first step in gene cloning is to isolate the DNA fragment or gene that contains the desired sequence to be cloned. This can be done by using different methods depending on the source and size of the DNA.

One common method is to use restriction enzymes, which are molecular scissors that cut DNA at specific recognition sites. By choosing the appropriate restriction enzymes, one can cut out a specific DNA fragment from a larger DNA molecule, such as a chromosome or a plasmid. The resulting DNA fragment will have sticky ends or blunt ends depending on the type of restriction enzyme used. Sticky ends are overhanging single-stranded regions that can base pair with complementary sticky ends from another DNA molecule. Blunt ends are flush-ended double-stranded regions that do not have any overhangs.

Another common method is to use polymerase chain reaction (PCR), which is a technique that amplifies a specific region of DNA using primers and a heat-stable DNA polymerase. By designing the primers to match the desired sequence, one can selectively amplify only the target DNA fragment from a complex mixture of DNA. The resulting DNA fragment will have blunt ends or sticky ends depending on the type of primers used.

A third method is to use reverse transcriptase, which is an enzyme that synthesizes a complementary DNA (cDNA) strand from an RNA template. This method is useful for isolating genes that are expressed as messenger RNA (mRNA) in cells. By using an oligo(dT) primer that binds to the poly(A) tail of mRNA, one can generate cDNA copies of all the mRNA molecules in a cell. Alternatively, by using a gene-specific primer that binds to a specific mRNA sequence, one can generate cDNA copies of only the target gene.

The isolated DNA fragment or gene can then be purified from other unwanted DNA molecules by using various techniques such as gel electrophoresis, column chromatography, or centrifugation. The purified DNA fragment or gene is ready to be inserted into a suitable vector for cloning.

A cloning vector is a DNA molecule that can carry a foreign DNA fragment into a host cell and replicate it. The choice of a cloning vector depends on several factors, such as:

• The size and source of the DNA fragment to be cloned
• The type and number of restriction sites on the vector and the fragment
• The host organism that will be used for transformation and propagation
• The purpose and application of the cloning experiment

Some of the common types of cloning vectors are:

• Plasmids: Circular, double-stranded DNA molecules that can replicate independently of the host chromosome in bacteria. They often carry genes for antibiotic resistance or other selectable markers. They can accommodate DNA fragments of up to 10 kb in size. Examples of plasmids are pBR322, pUC19, pBluescript, etc.
• Bacteriophages: Viruses that infect bacteria and integrate their DNA into the host chromosome. They can carry larger DNA fragments than plasmids, up to 25 kb in size. Examples of bacteriophages are lambda phage, M13 phage, etc.
• Cosmids: Hybrid vectors that combine the features of plasmids and bacteriophages. They have cos sites from lambda phage that allow packaging into viral particles and plasmid origins of replication and selectable markers. They can carry DNA fragments of up to 45 kb in size.
• Bacterial artificial chromosomes (BACs): Derived from the F plasmid of E. coli, they have a low copy number and a high stability. They can carry DNA fragments of up to 300 kb in size. Examples of BACs are pBAC108L, pBeloBAC11, etc.
• Yeast artificial chromosomes (YACs): Derived from the telomeres, centromeres and origins of replication of yeast chromosomes, they can replicate in yeast cells. They can carry DNA fragments of up to 1000 kb in size. Examples of YACs are pYAC3, pYAC4, etc.
• Mammalian artificial chromosomes (MACs): Synthetic chromosomes that can replicate in mammalian cells. They can carry DNA fragments of up to 10000 kb in size. Examples of MACs are human artificial chromosome (HAC), mouse artificial chromosome (MAC), etc.

The selection of a suitable cloning vector is crucial for the success of gene cloning. The vector should be compatible with the host cell, have a high transformation efficiency, have a high copy number or expression level, and have convenient features for cloning and screening.

All cloning vectors are carrier DNA molecules that can transport the gene of interest into a host cell and replicate it. However, not all DNA molecules can serve as cloning vectors. There are some essential characteristics that a cloning vector must have in order to be useful and efficient for gene cloning. These are:

• Self-replication: The vector must be able to replicate itself independently of the host chromosome. This ensures that the vector and the gene of interest are maintained and amplified in the host cell. Self-replicating vectors usually have an origin of replication (ori) that allows them to initiate and control their own replication.

• Unique restriction sites: The vector must have one or more unique restriction sites that can be recognized and cut by specific restriction enzymes. These sites are used to insert the gene of interest into the vector by creating compatible sticky or blunt ends. The restriction sites should not interfere with the function or replication of the vector.

• Selectable marker: The vector must have a selectable marker gene that can confer a distinct phenotype to the host cell. This allows the identification and selection of the transformed cells that carry the vector from the non-transformed cells that do not. The most common selectable markers are antibiotic resistance genes that enable the transformed cells to survive in the presence of a specific antibiotic.

• Cloning capacity: The vector must have a suitable size and structure to accommodate the gene of interest without affecting its stability or function. The cloning capacity depends on the type and complexity of the vector. Generally, plasmids can carry up to 10 kb of foreign DNA, bacteriophages can carry up to 25 kb, bacterial artificial chromosomes (BACs) can carry up to 300 kb, and yeast artificial chromosomes (YACs) can carry up to 1000 kb.

• Expression capability: The vector must have the necessary elements to allow the expression of the gene of interest in the host cell. These elements include a promoter, a ribosome binding site, a terminator, and sometimes an enhancer or a signal sequence. The expression capability depends on the compatibility of the vector and the host cell in terms of transcription and translation machinery.

These are some of the essential characteristics of cloning vectors that make them suitable for gene cloning. Depending on the purpose and application of gene cloning, different types of vectors may be chosen with different features and advantages.

Recombinant DNA (rDNA) is a hybrid DNA molecule that contains genetic material from different sources. To create recombinant DNA, the following steps are involved:

• The DNA fragment containing the gene of interest and the vector (a carrier DNA molecule that can replicate inside a host cell) are cut by the same restriction enzyme. This enzyme recognizes specific sequences of nucleotides and makes cuts at those sites, producing sticky ends or blunt ends on the DNA fragments.
• The DNA fragment and the vector are mixed together and annealed by base pairing of the complementary sticky ends or blunt ends. This forms a recombinant DNA molecule that has the gene of interest inserted into the vector.
• The recombinant DNA molecule is joined by the action of DNA ligase, an enzyme that seals the gaps between the fragments by forming phosphodiester bonds. This ensures that the recombinant DNA molecule is stable and intact.

The formation of recombinant DNA can be illustrated by the following diagram:

The recombinant DNA technology allows scientists to manipulate genes and create new combinations of genetic material that are not found in nature. By using recombinant DNA, scientists can isolate, characterize, and modify genes, as well as produce proteins and enzymes that have various applications in medicine, agriculture, and industry.

The recombinant vector is transformed into a suitable host cell, mostly a bacterial cell. This is done either for one or both of the following reasons:

• To replicate the recombinant DNA molecule in order to get multiple copies of the gene of interest (GI).
• To allow the expression of the GI such that it produces its needed protein product.

Some bacteria are naturally transformable; they take up the recombinant vector automatically. For example, Bacillus, Haemophilus, Helicobacter pylori, which are naturally competent .

Some other bacteria, on the other hand, require artificial methods for incorporation of DNA, such as:

• Calcium chloride treatment: The bacterial cells are treated with a cold solution of calcium chloride, which makes them permeable to DNA. Then they are exposed to a brief heat shock, which facilitates the uptake of DNA .
• Electroporation: The bacterial cells are exposed to a high-voltage electric pulse, which creates transient pores in their membranes, allowing the entry of DNA .
• Microinjection: The bacterial cells are injected with DNA using a fine glass needle under a microscope.
• Biolistics: The bacterial cells are bombarded with DNA-coated microscopic metal particles using a device called a gene gun.

The transformation process generates a mixed population of transformed and non-transformed host cells. The transformed cells need to be selected and identified in the next step.

10. Isolation of Recombinant Cells

The transformation process generates a mixed population of transformed and non-transformed host cells. The transformed cells are those that have taken up the recombinant vector, while the non-transformed cells are those that have not. To isolate the recombinant cells from the non-recombinant cells, a selection process is required. The selection process involves filtering the transformed host cells based on their ability to survive in a specific condition that the non-transformed cells cannot.

For isolation of recombinant cells, the marker gene of the plasmid vector is employed. A marker gene is a gene that confers a distinct phenotype to the host cell, such as resistance to an antibiotic or production of a color. The marker gene should be absent in the host cell before transformation, and present in the recombinant vector. For example, PBR322 plasmid vector contains two different marker genes: ampicillin resistant gene (ampR) and tetracycline resistant gene (tetR). When PstI restriction enzyme is used to cut the plasmid vector and insert the donor DNA fragment, it knocks out the ampR gene from the plasmid, so that the recombinant cell becomes sensitive to ampicillin.

To select for the recombinant cells, the transformed host cells are plated on a medium that contains both ampicillin and tetracycline. Only the recombinant cells that have taken up the plasmid vector with the tetR gene can survive on this medium, while the non-recombinant cells and the cells that have taken up the plasmid vector without the donor DNA fragment (which still have the ampR gene) will die. This way, only the recombinant cells are isolated from the mixed population.

Another example of a marker gene is lacZ gene, which encodes for β-galactosidase enzyme. This enzyme can cleave a synthetic substrate called X-gal and produce a blue color. The lacZ gene can be inserted into a plasmid vector along with a multiple cloning site (MCS), where different donor DNA fragments can be inserted. When a donor DNA fragment is inserted into the MCS, it disrupts the lacZ gene and renders it non-functional. The transformed host cells are then plated on a medium that contains X-gal. The recombinant cells that have taken up the plasmid vector with the disrupted lacZ gene will not produce β-galactosidase enzyme and will remain white, while the non-recombinant cells and the cells that have taken up the plasmid vector without the donor DNA fragment (which still have the functional lacZ gene) will produce β-galactosidase enzyme and turn blue. This way, only the recombinant cells are isolated from the mixed population.

The isolation of recombinant cells is an important step in gene cloning, as it ensures that only the desired clones are obtained and propagated for further analysis or application.

After the recombinant cells are isolated from the non-recombinant ones, they need to be grown and multiplied in a suitable culture medium. This step is important for two reasons:

• To obtain a large number of copies of the recombinant DNA molecule that carries the gene of interest.
• To allow the expression of the gene of interest and produce the desired protein product.

The culture medium should provide the optimal conditions for the growth and division of the host cells, such as temperature, pH, nutrients, oxygen, etc. Depending on the type of host cell, the culture medium may be solid or liquid. For example, bacterial cells are usually grown on agar plates or in liquid broth, while animal cells are grown in flasks or bioreactors containing a nutrient-rich solution.

The growth and multiplication of the host cells can be monitored by measuring parameters such as cell density, optical density, pH, etc. The host cells can also be induced to express the gene of interest by adding specific inducers or removing repressors from the culture medium. For example, some plasmid vectors have a lac operon promoter that can be activated by adding lactose or IPTG (isopropyl β-D-1-thiogalactopyranoside) to the medium.

The multiplication of the host cells results in the amplification of the recombinant DNA molecule and the production of the protein product. The amount and quality of the protein product can be assessed by using techniques such as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), Western blotting, ELISA (enzyme-linked immunosorbent assay), etc. The protein product can then be isolated and purified in the next step.

After the multiplication of the selected host cells, the product of interest can be isolated and purified from the culture medium. The product can be either the recombinant DNA molecule itself or the protein encoded by the gene of interest. The isolation and purification methods depend on the nature and location of the product.

• If the product is the recombinant DNA molecule, it can be extracted from the host cells by breaking them open and separating the DNA from other cellular components. This can be done by using physical methods (such as heat, sonication, or centrifugation) or chemical methods (such as detergents, enzymes, or salts). The recombinant DNA can then be purified by using techniques such as gel electrophoresis, column chromatography, or precipitation.

• If the product is a protein, it can be either secreted into the culture medium by the host cells or retained inside them. If the protein is secreted, it can be separated from the medium by using techniques such as filtration, centrifugation, or precipitation. If the protein is retained inside the cells, they have to be lysed first to release the protein. The protein can then be purified by using techniques such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, or electrophoresis.

The purity and quality of the product can be assessed by using methods such as spectrophotometry, SDS-PAGE, western blotting, or mass spectrometry. The purified product can then be stored or used for further applications.

Gene cloning has many applications in various fields of science and technology. Some of the major applications are:

• Biotechnology and medicine: Gene cloning can be used to produce recombinant proteins, enzymes, hormones, vaccines, antibodies and other therapeutic agents. For example, insulin, human growth hormone, erythropoietin, interferon and monoclonal antibodies are some of the products derived from gene cloning. Gene cloning can also be used to create transgenic animals and plants that have desirable traits or produce useful substances. For example, transgenic sheep that produce human clotting factor IX in their milk, transgenic mice that model human diseases, transgenic plants that resist pests or herbicides, etc. Gene cloning can also be used for gene therapy, which involves introducing a normal or modified gene into a patient`s cells to treat a genetic disorder or disease. For example, gene therapy has been used to treat severe combined immunodeficiency (SCID), hemophilia, cystic fibrosis, etc.
• Molecular biology and genetics: Gene cloning can be used to isolate and study specific genes and their functions. By cloning a gene, its nucleotide sequence can be determined and compared with other genes. Gene cloning can also be used to identify and analyze the regulatory sequences that control gene expression. Gene cloning can also be used to create mutations or modifications in a gene and study their effects on the gene product or phenotype. Gene cloning can also be used to create fusion genes that encode hybrid proteins with novel functions or properties. For example, green fluorescent protein (GFP) can be fused to a gene of interest and used as a marker to track its expression or localization in cells or tissues.
• Evolutionary biology and ecology: Gene cloning can be used to compare the genetic diversity and relationships among different species or populations. By cloning and sequencing genes from different organisms, phylogenetic trees can be constructed to show the evolutionary history and common ancestry of life forms. Gene cloning can also be used to study the adaptation and speciation of organisms in different environments. By cloning and analyzing genes that are involved in environmental responses or interactions, the molecular mechanisms of adaptation and speciation can be revealed. Gene cloning can also be used to conserve endangered species or restore extinct species by preserving or reintroducing their genetic material. For example, gene cloning has been used to clone endangered animals such as the giant panda, the black-footed ferret, the gaur, etc.

These are some of the applications of gene cloning that demonstrate its importance and potential in various fields of science and technology. Gene cloning is a powerful tool that can help us understand the molecular basis of life and improve its quality and diversity.

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