What is DNA cloning and why is it done?

Cloning refers to the synthetic production of mass quantities of DNA sequencesfor research or applied purposes. The cloning process starts with the identification and in silico construction of DNA sequences and regulatory elements that will be incorporated into plasmids and viral vectors. One goal of cloning is to facilitate studying the structure and function of genes and gene products, such as mRNA and protein. This research is critical to facilitate the development of diagnostic assays, vaccines and gene therapies.

Gene Cloning Vectors: Plasmids and Viruses Powering Life Sciences

Two main types of vectors are used for life sciences applications: plasmids and viruses.

Plasmids are circular cloning vectors that contain essential features like the origin of replication (ori), a selective gene (i.e., an antibiotic resistance gene) and a multiple cloning site (MCS) for recombinant gene insertions.

Viral vectors require mammalian cell hosts to enable their replication and packaging of DNA and regulatory element sequences.

Molecular cloning strategies often include using a reporter gene in a vector construct, such as bioluminescence genes, which reflect expression independently of selection pressure

Plasmid cloning vectors: Empowering Recombinant Gene Expression

Plasmids are ideal cloning vectors for containing and expressing recombinant gene insertions. Plasmids are small, circular pieces of DNA that exist independently in bacteria, such as Escherichia coli. Isolated plasmid DNA is easily manipulated with molecular biology techniques in vitro and packaged with regulatory coding sequences to initiate and regulate expression inside a host. A bacterial source, E. coli is widely used as a host for harboring plasmids that can be amplified and scaled to produce large quantities.

Plasmid cloning vectors include three essential features:

• An origin of replication (ORI) for amplification

• Selectable marker (i.e., antibiotic resistance gene)

• Multiple cloning sites (MCS) that allow gene insertions

The recombinant gene must include appropriate prokaryotic or eukaryotic regulatory sequences for upstream (5’) promoter activity and downstream (3’) termination sequences.

Additional gene sequences in DNA technology are available for plasmid vectors and include an eukaryotic expression vector such as the SV40 origin of replication that allows transient expression. Host-related homologous sequences embedded in a plasmid construct can assist site-specific vector integration into host genomes.

Multiple cloning sites (MCS) allow for the opening and resealing of a plasmid vector to facilitate the inclusion of a recombinant DNA sequence. Restriction enzymes cut the plasmid and recombinant DNA. The DNA ligase seals the plasmid back together for DNA cloning with plasmid vectors.

Viral vectors: Leveraging Viruses for Efficient Gene Delivery

The most common mammalian expression vectors are derived from viruses, such as adenoviruses, retroviruses and lentiviruses. These vectors typically contain strong promoters that drive high levels of gene expression, and they can be used to introduce genes into a wide variety of cell types.

Adeno-associated virus (AAV) is a popular choice of viral vector due to its high rates of gene incorporation and low abundance of viral sequences (i.e., inverted terminal repeats).

Although plasmids are the predominant cloning vector used in life sciences, a phage is another common type of protein expression vector. Phages are viruses that infect bacteria. Like plasmids, they can be used to express proteins in E. coli and other bacteria.

Cloning with Reporter Genes: : Illuminating Cellular Responses

Cloning vectors can include reporter genes to study the response of cells to various stimuli like growth factors, hormones and other extracellular signals. Researchers can use multiple reporter genes (multiplexing PCR) that encode various fluorescent proteins or enzymes to monitor the expression of different genes in the same cell. One reporter gene could be used to detect the expression of a gene of interest, while another could be used to distinguish a reference gene used for control purposes. Multiplexing provides a deeper understanding of the complexities involved in gene and cellular responses by monitoring and reporting on multiple parameters of the DNA fragments. This practice has applicability for drug discovery, bioengineering and gene therapy.

Reporter genes available for use in cloning experiments:

• Green Fluorescent Protein (GFP): This protein isolated from jellyfish emits green light when excited. GFP is frequently used as a marker protein to track the movement of other proteins or cells.
• Red Fluorescent Protein (RFP): Like GFP, RFP is derived from jellyfish and emits red light when excited. RFP is often used in conjunction with GFP to allow simultaneous tracking of two different proteins or cells.
• Yellow Fluorescent Protein (YFP): Derived from bacteria, YFP is another fluorescent protein that emits yellow light when excited.
• Luciferase: A reporter gene expressing luciferase is derived from fireflies and emits a blue-green light when luciferin is oxidized by molecular oxygen in the presence of ATP and luciferase. It is commonly used to detect gene expression and enzyme activity and for measuring cellular oxygen levels.
• Beta-Galactosidase: This enzyme catalyzes the breakdown of lactose into glucose and galactose. The presence of vectors expressing this enzyme in E. coli results in blue-colored colonies, while recombinant insertions interrupt beta-galactosidase activity and give rise to white colonies.

Future Applications for DNA Cloning: Pioneering Advances in Life Sciences

The applications of DNA cloning in the life sciences are diverse and constantly evolving. With the ongoing advancements in bioengineering and biotechnology, it is likely that new and innovative uses for cloning and the resulting DNA molecules will continue to emerge in the future.

Some of the future applications of cloning include:

1. Recombinant products: New cloning methods will continue to expand that help increase the quality and quantity of therapeutic genes for use in gene therapy and recombinant protein production.
2. Gene editing: Improvements to CRISPR technology and other gene editing tools combined with the ability to produce large quantities of complementary DNA and specific DNA sequences through advanced cloning methods will continue to accelerate new applications.
3. Synthetic biology: Cloning will continue to play an increasing role in enabling researchers to use artificial chromosomes and introduce new genetic information into synthetic biology systems to produce specific proteins or other biological products.

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