What is DNA cloning?
By definition, the general term “cloning” means obtaining multiples copies of something, let it be individuals of an animal species, or molecules. DNA cloning is a Molecular Biology technique that allows us to get multiple copies of identical sequence-specific double-stranded DNA fragments, for various purposes.
How is DNA cloning done?
To be able to clone DNA, we need the following elements:
- A source of the DNA of interest: normally, a bulk amount of the DNA of interest is necessary in the form of tissue, bacterial culture, etc. From this, the DNA is extracted through conventional extraction techniques.
- Plasmids: In order to be able to get multiple DNA copies, we need a self-replicating DNA molecule capable to carry the DNA fragment of interest. These DNA molecules are called plasmids, and they exist naturally in several microorganisms. These plasmids can be obtained in multiple copies per cell, being therefore possible to get substantial amounts of DNA to work with.
- Restriction enzymes: DNA cloning is possible because it can be cut at specific places (sequence-specific) thanks to a large set of proteins called “restriction enzymes” naturally appearing in many bacteria.
- Cells: We need living cells in which to introduce plasmids and amplify its amount. Normally bacteria are used, very convenient also thanks to its growth rate.
Basic steps of DNA cloning:
- Extraction of DNA thanks to DNA & RNA Extraction Kits.
- Restriction analysis and use of restriction enzymes to cut the suitable fragment of DNA to be cloned.
- Cutting the plasmid with the suitable restriction enzymes in order to be able to insert in it the desired fragment.
- Use of enzymatic DNA modifying enzymes (e.g. T4 DNA Ligase) to get a whole, circular plasmid-DNA insert (functional plasmid) capable of self-replication.
- Introduction of the plasmid into the cell by means of a technique called transformation.
- Culture of cells harboring the plasmid to amplify its amount.
- Selection of cells carrying the plasmid and the insert in culture plates by means of antibiotics and chromogenic substances, indicating the presence of a plasmid and a DNA fragment.
- Restriction or sequence analysis to check the cloned DNA sequence.
Where can cloning be used?
DNA cloning can be used in a wide series of applications, as for example:
- Protein production: If the cloned DNA codifies for a protein, large amounts of bacteria are grown in industrial fermenters, from which the desired protein can be purified. These proteins are normally biopharmaceuticals, like for example, insulin.
- Genetic analysis: Having a greater amount of DNA makes possible to analyze genetic material in detail, to study DNA transcription, translation of transcripts, DNA profiling in search of specific sequences (including forensic and criminal investigations), amplification of very small amounts of genetic material, etc.
- Gene therapy: Specific DNA sequences can be used to produce proteins directly into cells in which defective genes are present causing a genetic disease. This set of techniques is referred to as “gene therapy” and allows treating previously untreatable diseases.
What are the best tools to clone in a lab?
We highly recommend pSpark® Blunt-end DNA Cloning family vectors. The vectors have a proven track record as the most efficient, powerful and easy way to obtain the best results. Nine different vectors are available for Blunt-end, TA or Universal DNA Cloning that adapts perfectly to your research to get the best results.