Subcloning is a crucial technique in the field of molecular biology that allows the transfer of known fragments of target DNA from one vector to another new vector. It can be used to create new gene structures, study changes in gene function, and develop new gene therapies. In addition, it can be used to construct gene expression vectors to express target genes in specific cells or tissues.

Applications of Subcloning Technology

Easy Sequencing: when DNA is on a small plasmid, sequencing can be done quickly and efficiently. Existing high-throughput sequencing platforms are utilized to quickly and accurately read these short sequences. This approach not only improves sequencing efficiency, but also reduces costs and makes it easier and more precise to study specific genes or genetic regions.

Structural Analysis: Target DNA obtained through subcloning methods can be used for detailed physicochemical property analysis, including but not limited to the application of advanced techniques such as X-ray crystallography, nuclear magnetic resonance (NMR), and others. This helps to gain insight into the specific three-dimensional conformation of the DNA and the way it interacts with proteins and small molecules, revealing its potential function.

Functional Studies: Use of homologous recombination pathways in yeast coupled with specific linkers to perform secondary cloning of target regions of large DNA fragments (e.g. from bacterial artificial chromosomes) for functional analysis of complex genomes. For example, the study of multigene family regulatory networks associated with complex traits in plants or animals.

Disease Modeling: mice or other model organisms capable of mimicking mutations associated with human genetic diseases through subcloning, which can be characterized by mutations typical of human genetic diseases. Such transgenic models are useful for understanding the pathogenesis of diseases, testing the effects of new drugs, and optimizing therapeutic regimens, thereby accelerating the drug discovery process.

Therapeutic Applications: subcloning allows precise control of which sequences are expressed and can be used to develop nucleic acid-based technologies (e.g., RNA interference therapy). Design nucleic acid drugs that specifically target certain disease-causing genes, such as siRNAs (small interfering RNAs). By carefully selecting and expressing specific sequences, the production of harmful gene products can be effectively inhibited for therapeutic purposes. Such therapies have already shown great potential in the field of cancer treatment and are expected to be applied to the treatment of more types of hereditary diseases in the future.

SubcloningExperiment Process and Precautions

1. Preparation of target DNA fragments and vectors

Ensure the integrity of the target DNA fragments to avoid breakage or degradation during extraction, purification or processing. Adjust the concentration and purity of the target DNA fragments to minimize non-specific binding in the ligation reaction.

2. Ligation between the target DNA fragment and the vector

Select the appropriate type of vector (plasmid, viral vector, etc.) and use the appropriate restriction endonuclease to cleave the vector to produce sticky or flat ends matching the target DNA fragments, and use alkaline phosphatase to dephosphorylate the vector.

3. Transformation of ligation products

Select a highly efficient ligase, such as T4 DNA ligase, to ensure the correct ligation between the target DNA fragment and the vector. Optimize the conditions of the ligation reaction (temperature, time and amount of ligase used, etc.) to improve the ligation efficiency. The presence and size of the ligation product can be verified by electrophoresis or other methods to ensure the success of the ligation reaction.

4. Transformation and positive clone screening

Choose the appropriate transformation method according to the experimental needs, such as thermal or electrical transformation. Antibiotic resistance screening, PCR screening or sequencing verification can be used to screen out clones containing the correct insert fragments.

Synbio Technologies | PCR subcloning

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.

PCR cloning and subcloning workflow

Reference

1. Džunková Mária, D’Auria Giuseppe, Pérez-Villarroya David, et al. Hybrid Sequencing Approach Applied to Human Fecal Metagenomic Clone Libraries Revealed Clones with Potential Biotechnological Applications[J]. PLoS ONE,2012,7(10):e47654-e47654.

2. Raymond Christopher K., Sims Elizabeth H., Olson Maynard V.. Linker-Mediated Recombinational Subcloning of Large DNA Fragments Using Yeast[J]. Genome Research,2002,12(1):190-197.

3. Dang H.X., White B.S., Foltz S.M., et al. ClonEvol: clonal ordering and visualization in cancer sequencing[J]. Annals of Oncology,2017,28(12):3076-3082.

4. Bohula Erin A., Salisbury Amanda J., Sohail Muhammad, et al. The Efficacy of Small Interfering RNAs Targeted to the Type 1 Insulin-like Growth Factor Receptor (IGF1R) Is Influenced by Secondary Structure in the IGF1R Transcript[J]. Journal of Biological Chemistry,2003,278(18):15991-15997.