Gene Cloning

Gene cloning is a technique used in genetic engineering to create copies of specific genes or DNA sequences. It allows scientists to isolate and amplify genes for research purposes, drug development, and improving crop yields.

Gene cloning is a procedure that permits researchers to copy and focus on one specific gene rather than dealing with the entire genome. It has become an indispensable tool in modern biotechnology because it produces similar copies of genes.

Cloning is a method of isolating and amplifying genes by cutting out desired sections from DNA using restriction enzymes and inserting them into another piece called a vector through a ligation reaction. The technique known as gene cloning, DNA cloning, molecular cloning or recombinant DNA technology all refer to the same thing and are used interchangeably by scientists depending on their preferences or fields of study. This article demonstrates gene cloning and its significant details.

Gene Cloning Requirements (Cell-Based)

• A DNA fragment that contains desired genes for the purpose of cloning.
• Ligase, as well as the restriction enzyme.
• Vectors transport, sustain and reproduce cloned genes in host cells.
• A host cell that can replicate recombinant DNA.

Principles and Steps in Gene Cloning

To clone a gene, it is inserted into a vector which acts as a vehicle to transport the gene into a host cell where the vector multiplies and creates similar copies of itself and the gene. Bacteria are commonly utilised as host cells for cloning, however other living cells can also be utilised.

During cell division, recombinant DNA molecules are passed down and replicated in the host cells. After numerous divisions, a clone of identical cells is produced with one or more copies of the recombinant DNA molecule, resulting in gene cloning.

Steps Involved in Gene Cloning

There are seven significant steps in gene cloning which are as follows.

1. Isolate DNA Fragments for Cloning

• To clone a gene, it must first be isolated. It involves identifying the specific fragment of DNA that codes for the desired protein or hormone, such as insulin.
• The gene can be obtained using a restriction endonuclease enzyme that cuts the DNA at specific sites, producing blunt or sticky ends.
• Reverse transcriptase can create a complementary DNA strand of a desired gene using its mRNA.

2. The Process of Creating Recombinant DNA by Inserting isolated DNA into a Vector

• Vectors are molecules that can transport a desired gene into a host and replicate it alongside the gene to create multiple copies.
• When choosing a cloning vector, it's essential to consider the size of the insert and its intended use. Plus, different vectors have different restrictions on the size of insert they can carry.
• Cloning vectors include plasmids, bacteriophages, BACs, YACs and MACs.
• Cloning vectors mainly utilised plasmids and bacteriophages such as phage λ.

3. Introducing Recombinant DNA into an Organism known as the Host

Cloning vectors are carrier DNA molecules with common features.

• It must self-replicate within the host cell.
• It needs to have a distinct restriction site for RE enzymes.
• It is vital to ensure that the introduction of donor DNA does not disrupt the replication ability of the vector.
• The recombinant cell needs a marker gene for identification (typically an antibiotic-resistance gene not found in the host cell).
• The host cell should easily isolate them.

4. The Process of Selecting transformed Host Cells and Identifying the Clone that contains a Specific Gene

• The plasmid vector is opened using the same restriction enzyme used to isolate the donor DNA fragment.
• A donor DNA fragment and plasmid vector are combined.
• DNA ligase facilitates the base pairing of donor DNA fragments and plasmid vectors.
• The DNA molecule created is a combination of two different molecules, known as recombination in genetics.
• The technology of combining DNA from diverse sources is known as recombinant DNA technology, creating a hybrid molecule which is known as a recombinant DNA molecule.

5. The Expression of a Gene in a Host Organism via Multiplication

The recombinant vector is mostly transformed into a suitable host cell, usually a bacterial cell.

This is done for the following reasons:

• To make multiple copies of the GI, replicate the recombinant DNA molecule.
• Enable GI expression for required protein production.
• Certain bacteria have the ability to naturally take in recombinant vectors without external assistance. Examples of naturally competent bacteria include Bacillus, Haemophilus, and Helicobacter pylori.
• Certain bacteria need artificial methods like Ca++ ion treatment and electroporation for incorporation.

6. Isolation of Multiple Gene Copies/Proteins

• During the transformation process, both transformed, and non-transformed host cells are produced.
• The process of selection involves only filtering the transformed host cells.
• A marker gene of a plasmid vector is used to separate recombinant cells from non-recombinant cells.
• For instance, The PBR322 plasmid vector has two marker genes, Ampicillin resistant and Tetracycline resistant. Using the pst1 RE enzyme removes the Ampicillin-resistant gene from the plasmid, making recombinant cells sensitive to Ampicillin.

7. Purification of Genes/Protein

• After host cells have been transformed and screened, it is important to provide them with optimal conditions for growth and replication.
• After the transformation process, the modified host cells are added to new culture media during this step.
• Next, isolate the multiplied GI with the attached vector or encoded protein.
• Purify the isolated gene copy or protein.
• During this phase, the host cells undergo multiple rounds of division while also replicating the recombinant DNA they contain.
• To obtain the desired product using a vector, favourable conditions must be provided for gene expression. Replicating the host cell is sufficient for obtaining multiple copies of the gene of interest.

Gene Cloning Applications

1. Gene Function: Gene function is a crucial aspect of DNA and gene analysis. Cloning genes allow for the determination of their existence or non-existence.

The recombinant plasmid can be used to establish the purpose of a gene, as well as for gene knockout, knockdown and transgene creation. PCR is an effective, quick and economical technique that has replaced conventional methods for cloning genes by increasing or synthesizing them with Taq DNA polymerase.

2. Gene Structure: Cloning can be utilized to identify and sequence the structure of a particular gene, allowing for analysis of variations in its sequence.

3. Constructing Genetically Modified Organisms: Gene cloning is a popular technique for constructing genetically modified organisms through recombinant DNA. It involves inserting a transgene into plants, bacteria or mice to modify their characteristics and phenotypes, as well as create disease-related models.

4. Gene Therapy: Gene therapy uses gene cloning to replace or repair damaged DNA sequences or genes with healthy versions, providing a potential treatment option for genetic diseases.

5. Mutational Studies: The polymerase chain reaction technique has replaced gene cloning for mutational studies and identification, as it is faster and cheaper. It can also be used for site-directed mutagenesis research.

The isolation and analysis of genes can reveal their nucleotide sequence, control sequences, and functional properties. This information enables the identification of mutations related to diseases and the engineering of organisms for specific purposes, such as insulin production or insect resistance.

6. Biopharmaceuticals: Gene cloning is utilized for the creation of synthetic as well as recombinant proteins, including insulin and growth factors. This procedure is considered innovative in the biopharmaceuticals field.

Gene Cloning Limitations

In the 80s, DNA cloning was a significant discovery, but it has limitations and drawbacks.

• The current method is slow, taking 3-4 days to produce results due to time-consuming steps such as culturing and restriction digestion.
• The technique is expensive and difficult, and there are concerns about contamination.
• The experiment has low accuracy and yield.

Conclusion

Gene cloning is a process of making copies of DNA, but it has limitations. The discovery of the Polymerase chain reaction in 1983 overcame these limitations and provided a rapid, cost-effective, accurate and high-yield technique for gene cloning.

PCR is a powerful tool in genetics and genomics as it saves time, and money and provides ease in experiments by producing more DNA copies than conventional gene cloning techniques.

FAQs

1. What is the Main Purpose of Gene Cloning?

Cloning is the process of replicating DNA to produce identical copies of specific genes from cells of other organisms. This technology helps researchers study how genes influence characteristics and grow large quantities of genetically modified crops that resist pests.

2. What is the role of DNA Ligases in Gene Cloning?

DNA ligases are crucial for gene cloning as they facilitate the bonding of DNA strands during cell division through a process known as ligation.

3. Why use PCR before Gene Cloning in Cells?

PCR is a technique used to amplify DNA templates for analysis by copying large amounts of DNA from one sample into another through repeated cycles of heating and cooling at specific temperatures.