What is Cloning?
"The terms recombinant DNA technology, DNA cloning, molecular cloning, or gene cloning all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid (cloning vector). The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today." (from the Cloning Fact Sheet more info.)
Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis.
How Does Molecular Cloning Work?
Cloning of any DNA sequence involves the introduction of a foreign piece of DNA into an extrachromosomal element (cloning vector) of an organism which then produces copies of the vector as it replicates itself, thereby amplifying the DNA of interest. The whole process can be summarized in the following steps: fragmentation, ligation, transfection, screening/selection, and conformation of insert.
A cloning vector is simply a DNA molecule that can be inserted into a host cell and replicates inside the host (bacteria or yeast), producing many copies of itself and the insert DNA. All cloning vectors contain -
- A sequence that allows for the propagation of itself in the host.
- An insertion site for the foreign DNA - also called a multiple cloning site that can be cut by several restriction enzymes.
- A method for selection of the host cells that contain the insert DNA of interest. This is most often done through the use of selectable markers for drug resistance.
Types of Cloning Vectors:
- Plasmids - extrachromosomal circular DNA that autonomously replicates itself in a bacterial cell. Plasmids can be high or low copy numbers with an insert limit of about 10,000 base pairs (10 Kb)
- Phage - viruses that infect bacteria (usually derivatives of lambda bacteriophage). They are normally linear DNA molecules with regions that can be replaced without disrupting their ability to direct their replication by their bacterial hosts - insert limit of about 20 Kb
- Cosmids - circular extrachromosomal elements that combine elements of plasmids and phage - insert limit of 35-50 Kb.
- Bacterial Artificial Chromosomes (BAC's) based on mini-F plasmids - insert limits of 75-300 Kb.
- Yeast Artificial Chromosomes (YAC's) - artificial yeast chromosomes that contain telomeres, an origin of replication, a yeast centromere, and a selectable marker for identification in yeast cells - insert limits of 100-1000 kb.
- amplifying 16s RNA genes to identify individual microbial species
- create cDNA libraries to determine what genes are being expressed at a particular time
- create genomic libraries in order to sequence organisms
- create multiple clone libraries in order to conduct metagenomic studies
How to Clone DNA Fragments -
- Fragmentation - Initially, the DNA of interest needs to be fragmented to provide a relevant DNA segment of suitable size. Preparation of DNA fragments for cloning is frequently achieved by means of PCR, but it may also be accomplished by restriction enzyme digestion and sometimes fractionation by gel electrophoresis.
- Ligation - Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase.
- Transfection - Following ligation the vector with the insert of interest is transfected into cells. Most commonly electroporation is employed, although a number of alternative techniques are available, such as chemical sensitization of cells.
- Screening and Selection - Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is need to identify the cell colonies that have been successfully transfected with the vector construct containing the desired insertion sequence. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain color selection markers which provide blue/white screening on X-gal medium.
- Conformation - Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and DNA sequencing.