Introduction of Molecular Cloning
Cloning at the molecular level is a standard practice in laboratories dedicated to molecular biology and biotechnology. It is a helpful tool for producing protein, studying genes, modifying genes, reintroducing modified genes into their natural hosts or hosts, and studying how genes interact with other genes. You can carry out the process of molecular cloning by inserting a segment of DNA into a plasmid, also known as recombinant DNA, in order to produce more similar recombinant DNA in an organism that is still alive. Before beginning cloning, it is important to examine a number of factors, including the accessibility of your cloning material, the purpose of your research (whether it be to sequence a DNA fragment or generate a protein), and the degree of complexity required to carry out your technique. The steps involved in molecular cloning are as follows:
The extraction of DNA and its preparation: In order to extract DNA, the cells must first be lysed, and then the DNA must be solubilized. Next, either chemical or enzymatic procedures are used to remove any remaining polysaccharides, triglycerides, RNA, or proteins. The process of DNA extraction may be broken down into five primary phases that remain the same across the various chemistries that can be used to purify DNA. 1) the destruction of the cellular structure in order to produce a lysate; 2) the isolation of the solubilized DNA from the cellular debris and other undissolved material; 3) the adhesion of the DNA of interest to a detoxification matrix; 4) the washing of polypeptides and other residues away from matrix and 5) the elution of the DNA.
DNA ligation refers to the process in which two DNA molecules are joined together by the enzyme known as DNA ligase. In an ATP-dependent procedure, DNA ligase acts as a catalyst for the production of two covalently phosphodiesters between both the 3' hydroxyl group of one nucleotide as well as the 5' phosphate molecule of other nucleotide. These linkages are phosphodiester bonds. In addition, a gene-selective marker for genetic tolerance will be inserted at this stage.
The next step in the cloning process is called transformation, and it occurs after a DNA fragment has been inserted into a plasmid vector (a DNA molecule that can carry a DNA insert to produce recombinant DNA and simulate it in a specific host). Plasmid vectors are used in the process of cloning because they allow for the generation of recombinant DNA. During this phase of the transformation process, either a chemical process or electroporation is used to transfer the recombinant DNA into a competent cell (figure below). Competent cells are cells that are momentarily accessible to extracellular DNA. Competent cells make up the body's immune system. Escherichia coli, as well as Saccharomyces cerevisiae, are two examples of host organisms that are frequently utilized in research facilities.
Figure. Electroporation process. Source
Plate streaking and selecting antibiotics Streak plates are created by progressively diluting an inoculated bacteria and yeast spread out over the top of a medium that has been hardened using agar in a Petri dish. As a consequence of this, some of the colonists on the plate grow in a manner that is sufficiently distinct from one another. The procedure's goal is to produce single colonies that are pure and isolated from one another. A growth medium that contains a selective agent is referred to as a selective medium. This type of medium is used to cultivate transformed hosts. If you choose the antibiotic collection for the cloning process, the growth medium you use absolutely needs to include antibiotics. Kanamycin, ampicillin, and chloramphenicol are the three antibiotics that are utilized for selecting the most frequently.
Following this step, the actual process of gene expression takes place within the growth medium. The process by which new amplicons are produced in response to external stimuli is referred to as inducible gene expression. A significant portion of the production of new amplicons occurs as a result of rapid activation of transcriptional activation. Recent years have seen an increase in the number of cloning techniques that make use of green fluorescent protein, also known as GFP, as a reporter gene. It is essential to investigate the ways in which cellular processes adapt and change in living cells. In addition to that, it has a low potential for toxicity, a high degree of stability, and non-invasive identification. Because of its capacity to generate green light without the need for any additional expensive equipment or the incorporation of any additional external factors, it is, therefore, best suited for the role of a reporter gene.
Why is molecular cloning a confusing topic for students?
1. Theoretical subject
The field of molecular cloning is advanced, and there are many misconceptions about it. Seeing as how this subject can only be taught through the use of theories and textbooks, its practical use is limited. Students' mental pictures of the subject matter may get confused if they solely engage in that style of learning.
2. Weak molecular biology background
Cloning at the molecular level is a complicated subject. There is a possibility that students are unfamiliar with fundamental concepts and terms, such as recombination, plasmids, vectors, replications, and PCR, amongst others. They may have difficulties comprehending the material because they are unfamiliar with such phrases. Because of this, students could find that the subject no longer holds their interest.
3. The complex mechanism
The process of molecular cloning is a multi-step one that requires the methodical arrangement of a number of different procedures. It is possible for students to feel overwhelmed when learning the specifics of these processes and the reasoning behind them.
Five ways to conceptualize Molecular Cloning among students?
1. Provide them facts
Students can effectively have a solid understanding of the issue and build their interest by receiving factual information about it. Some facts regarding cloning include the following:
Over the course of the past half-century, researchers have successfully cloned a wide variety of animal species.
The first fish to be replicated was an Asian goldfish in 1963 by a Chinese scientist named Tong Dizhou.
· In 1986, a house mouse was cloned in the Soviet Union using an embryo cell.
The cloning of an actual living human being has never been proven to have taken place, despite several claims to the contrary.
Cloning humans has been outlawed in a number of nations due to social, ethical, and religious concerns it raises.
Cloning is a technique that has the potential to be utilized in the future to help save endangered animal populations. It is likely that it will be possible to revive formerly extinct species in the near future.
2. Difference between terminologies
Students must distinguish between key terms such "plasmid vs. What is the mechanism of DNA damage and repair? Two separate kinds of double-stranded DNA molecules, vector, and plasmid, each serve a unique purpose in the cell. The fundamental distinction between plasmids and vectors is that the former are extra-chromosomal components found mostly in bacterial cells, while the latter are carriers of foreign DNA molecules into other cells.
The removal of the affected bases is followed by synthesis and secretion of the excision region, which is how most DNA damage is healed. Nevertheless, some DNA lesions can be directly reversed in order to be repaired, which may be a more effective strategy to handle some kinds of DNA damage that are common.
3. Awareness of Pros and Cons of Cloning
Explain to the students why cloning is being used in the current application. There are many different scientific applications for therapeutic cloning. Among these are non-reproductive cloning techniques and stem-cell research. Therapeutic cloning has a lot of potentials to reduce suffering and increase scientific understanding.
Cloning is extremely expensive, though; any advancements won't happen for decades and will only help the powerful and wealthy. Cloning should be abandoned in favor of tackling pressing issues like AIDS, malaria, or tuberculosis that affect millions of primarily underprivileged people globally.
4. Awareness of ethical issues
Cloning, in its third iteration, has some concerns about the immaterial, about interfering with Nature, and about God. Richard Seed used this as his theological justification for why it is okay for humans to control their reproductive and destiny processes. This very justification is used by many nations to forbid human cloning. Between the scientific establishment and numerous institutions and individuals around the world, bioethics continues to be a contentious issue.
5. Use virtual lab simulations
The topic of cloning can be complicated and vast for students. Labster offers a solution to this issue by providing 3D simulations of molecular cloning. Your lecture presentation and lab management activities will be made easier by Labster's virtual laboratory simulations. Given that students have access to visual alternatives, you might be able to provide arguments that are more thought-provoking. You will learn how to put together an expression vector incorporating RAD52 and GFP in the molecular cloning lab. The goal is to use Doxycycline to regulate RAD52's expression level and evaluate that level by looking at the GFP signal. Our virtual classroom uses this dynamic, immersive education methodology to reinforce key concepts for emerging scientists. Use Labster's Molecular Cloning simulation of molecular cloning in your forthcoming class to fill in the gaps in the accessible instructional materials and tackle the absence of aesthetically appealing video graphical alternatives in the educational area.