Molecular cloning technology operates at the molecular level. It is used to isolate specific DNA fragments from donor organisms and then insert them into suitable cloning vectors through methods such as in vitro recombination, transformation, and transduction. The recombinant cloning vectors are introduced into appropriate hosts for replication and amplification. Finally, the desired cloning vectors are screened and isolated to obtain multiple copies of the target DNA, thereby achieving the amplification of the target gene. It can be applied to research in protein expression, virus packaging, gene therapy, cellular therapy, mRNA vaccines, RNA interference, cellular functions, and more.

The Technical Process of Molecular Cloning

Selection of the Target Gene

• Target DNA Selection: The process starts by identifying and selecting the gene or DNA sequence you wish to clone. This gene can be from any source, such as a prokaryotic or eukaryotic organism.

• DNA Extraction: The source DNA (genomic, plasmid, or cDNA) is extracted from the cells using standard DNA isolation techniques.

Vector Linearization: The vector is treated with restriction enzymes (endonucleases) to create sticky or blunt ends for ligation with the gene of interest. The selection of restriction enzymes depends on the cloning strategy (e.g., compatible restriction sites in both the vector and insert).

Ligation Reactions: The linearized vector and the target gene were mixed under the action of DNA ligase. If the end is compatible, the connection efficiency is high. The linker mixture is usually cultured at room temperature or 16 ° C for several hours.

Transformation into Host Cells: After ligation, the recombinant plasmid is introduced into bacterial cells via transformation. The cells are made "competent" (able to take up foreign DNA) by treatment with calcium chloride or by electroporation. Then, the cells are plated onto an agar plate containing a selective agent (e.g., antibiotics).

Selection and Screening: If the plasmid contains a selectable marker, only those bacteria that have successfully taken up the plasmid will survive on the selective medium. Colonies that grow on selective plates are picked and cultured for further analysis. These colonies are often screened for the presence of the recombinant DNA insert.

Research Areas of Molecular Cloning

Gene Cloning and Expression: Molecular cloning enables the amplification and expression of target genes. This technology is crucial for studying and understanding gene functions and developing new gene therapies and drugs. For instance, it allows for the study of interactions between gene expression products, protein structures, and functions.

Virus Packaging and Gene Therapy: Through molecular cloning, scientists can create recombinant vectors containing specific viral genes and then introduce them into host cells to produce viral particles. This technology is widely used in vaccine development and gene therapy.

Cellular Therapy and Gene Modification: Molecular cloning allows scientists to modify cell genomes, thereby altering cell characteristics and functions. This technology has significant applications in studying disease mechanisms, developing new cellular therapies, and producing cell lines with specific functions. For example, technologies such as CRISPR-Cas9 can achieve site-specific knockout, insertion, or replacement of specific genes; or CAR-T cells can be constructed to treat tumors, and stem cells can be used to treat spinal cord injuries.

Proteomics and Biomolecule Research: Molecular cloning can be used to create recombinant vectors containing genes encoding specific proteins or biomolecules, which are then expressed and purified in host cells. This technology is essential for studying protein structures and functions and developing new drugs and therapies.

Gene Knockout and Functional Research: Through molecular cloning, recombinant vectors containing specific gene knockout sequences can be created and introduced into cells to delete the target gene. This technology is widely used to study gene functions and determine the roles of specific genes in cellular biology. For example, knocking out specific genes in mice or other model organisms can help study their effects on growth, development, and metabolism.

Drug Screening and Research: Molecular cloning can be used to construct drug screening models to rapidly identify drugs with specific mechanisms of action. For instance, mutant cell lines or model organisms can be created to study drug mechanisms and effects.

Agriculture and Animal Breeding: Molecular cloning can be used to cultivate transgenic crops and animals, improving yield, resistance, and the quality of agricultural products. Additionally, it can be applied in animal breeding to achieve combinations of desirable genetic traits.

PCR Cloning & Subcloning |Synbio Technologies

Molecular cloning, as a commonly used recombinant technology, is widely used in genetic engineering, agricultural production, biopharmaceuticals, etc. Synbio Technologies, leveraging the integration of DNA synthesis tools and cloning technologies, offers comprehensive PCR cloning and subcloning services. Our services are capable of cloning the target gene into any desired location of the vector, fulfilling your specific design requirements. For subcloning the same gene fragment into different vectors, we also provide efficient services to ensure the smooth progress of your downstream experiments.

References

[1] Lessard JC. Molecular cloning. Methods Enzymol. 2013;529:85-98.