DNA cloning represents a molecular biology method that consists of isolation identical individual genes or segments of a specific DNA fragment and then transferring the fragment into a plasmid vector. Being introduced in the plasmid vector, the DNA fragment can be then amplified, sequenced, stored and used for gene expression.
The main steps of this experiment are finding the location of the DNA region of interest, followed by cutting that distinctive molecule and also purify it in order to avoid the presence of undigested substances that may alter the experiment. In this case, the gene of interest is EZH2, or Enhancer of zeste homolog 2, which is a subunit of PRC2 (polycomb repressive complex 2) and is used to repress expression of the genes. – https://www.ncbi.nlm.nih.gov/gene/2146
The plasmid vector, a circular piece of DNA can be amplified in E.coli and later manipulated, is pBluescript.
Another vital step is to insert the piece of DNA onto a cloning vector which will allow the EZH2 (the cloned sequence) to be transported into a new host (another plasmid vector) and aid further steps, such as transcription and translation of the fused DNA fragment. -https://khairulanam.files.wordpress.com/2010/08/cloning-ii.pdf
Digestion of the DNA is also an important step because after cutting both the DNA and the pBluescript (plasmid vector) with the same restriction enzyme (EcoRI), their “sticky ends” should be compatible, which will make the next step, ligation, more efficient and those 2 molecules should fuse together. In order to perform this step, the pBluescript has to be linearized so that the both DNA molecules can have compatible ends to be able to join together. Also, if both vector and insert are digested with the same restriction enzyme, EcoRI, their single stranded overhands will have complementary sequences and, therefore, the ligation will occur easier (?).
To improve this temporary fusion, phosphodiester (covalent) bonds need to be formed in a new process called ligation. Ligation is the step where, in the presence of DNA ligase, the ends of the 2 linear molecules of DNA are joined together to form a single intact DNA molecule. The most commonly used DNA ligase and the one also used in this experiment is T4 DNA ligase. For this step to occur, T4 DNA ligase needs ATP and magnesium ions for energy and acticity.
Once the ligation mixture was fused into 1 unbroken DNA molecule, the new molecule is incorporated into the bacterial cells (E.coli), in order to be replicated and expressed the gene later. This process is called transformation.
A his-tag, or a polyhistidine-tag, is a 6 histidine residues of amino acid motifs at either the N or C terminus of any recombinant protein and it’s purpose is to isolate that protein. In this case, the his-tag was attached at the C terminus. (?)
The aim of the experiment is to perform a cloning of the gene, in this case EZH2, by fusing it with the his-tag, the recombinant protein.