Cloning is a technique to make copies of the genetic material of living organisms. In this advanced era, scientists can produce genes, tissues, and even a whole organism by cloning. There are several cloning methods but Uracil based excision cloning or USER is ligase free cloning method.
Uracil based excision cloning (USER) is a technique in which fragments of DNA are used as vectors to produce stable hybridized products. The USER cloning method consists of USER cloning, fusion, and USER cassette-free fusion. It helps to combine several PCR fragments, changes the nucleotide sequence, and does directional cloning.
USER (Uracil Specific Excision Reagent) is an enzyme that contains Uracil DNA glycosylase and DNA glycosylase-lyase. This method needs a short identity between the sequences, so the adjacent sequence can overlap with each other. At the 3’ end of the overlap, the presence of uracil residue is also necessary for cloning.
In Uracl based excision cloning (USER) technique, the target DNA and the vector fragment are produced by PCR. The primer contains a single deoxyuracil residue (dU) with 3’ overhanging. It can be designed to insert, delete or substitute any nucleotide. Then, the primer can help to amplify the target DNA and the vector fragment. It creates a single nucleotide gap with each location of deoxyuracil residue (dU) and produced a linearized vector. In this way, multiple DNA fragments can be cloned by a single experiment.
Uracil based excision cloning (USER) is beneficial because it is a simple cloning technique. There is no requirement for ligase enzymes which makes the USER method easier.
For some readers, Uracil based excision cloning (USER) can be difficult to understand. The process, mechanism, the role of uracil, and functions of enzymes can be tricky to read. But there are some ways that can help to make this topic more interesting for students.
There are top three reasons why students find Uracil based cloning (USER) difficult to learn.
There are two types of primer design; the 5’ overhang region and the Binding region. The 5’ overhang region can be responsible for the production of 3’ cohesive overhangs that help to ligate the fragments of DNA. The main three rules for primer overhang should be difficult to remember because a student needs to remember the length, temperature, and DNA sequence design.
As we cannot see the cloning process with the naked eye, it feels abstract. It always requires a microscope and a trained person to see the DNA and the whole cloning process. Therefore, some students may find it hard to believe that Uracil based excision cloning (USER) is taking place as mentioned in the textbook.
Uracil based excision cloning (USER) comprises three enzymes. These are high fidelity DNA polymerase, Endonuclease VIII, and Uracil DNA glycosylase. It is difficult for students to remember the names and the action of these enzymes in the USER cling method. You can say that the study material of Uracil based excision cloning (USER) is heavy and complicated.
Since knowing the difficulties that students may face during the learning of Uracil based excision cloning (USER), here are 5 ways to make USER a more interesting topic to understand. If you adopt these five ways, it will be easier for you to understand the Uracil based excision cloning (USER).
The scientific history span of cloning is more than 100 years. Uracil is one of the nucleobases of RNA and binds to adenine by two hydrogen bonds. In 1900, Uracil was discovered by an Italian serologist Alberto Ascoli. He used herring sperms and isolate the Uracil by process of hydrolysis. Later, it was also obtained from the spleen, thymus, and wheat germ.
In 1901, the structure of Uracil synthesis was proven. In this structure, hydrolysis of ribonucleic acid gives nucleotide known as uridylic acid. The next step is the removal of phosphate then it undergoes hydrolysis to finally produce Uracil.
The concept of Uracil based excision cloning (USER) was perceived in the 1990s. The result of Uracil based excision cloning was first obtained in 2003. Now, scientists use Uracil for cloning purposes which makes the cloning methods simpler.
It is always recommended to first introduce the basic concept of the topic. For instance, in this topic, students should know about Uracil and the role of Uracil in gene cloning. Without basic knowledge about Uracil, it can be difficult for students to understand Uracil based excision cloning.
RNA (Ribonucleic acid) is a nucleic acid that is present in all living organisms. It consists of four nucleotides; Uracil is one of them. In RNA, Uracil always pairs with Adenine with the help of two hydrogen bonds. Uracil belongs to pyrimidine nucleobase and the chemical formula of Uracil is C4H4N2O2.
Uracil based excision cloning is a ligase-free method that uses the insertion of PCR fragments into cloning adapted plasmids. There are four different protocols for Uracil base excision cloning (USER) cloning.
First protocol for simple manipulation
Second for Plasmid based multigene assembly in E.coli
Third protocol for integration of genes into the genome
Last protocol for pathway assembly and multigene expression optimization
When a topic is difficult to understand just like Uracil based excision cloning, then it is important to use color diagrams. Visual diagrams can make learning easier and simple. Color diagrams can enhance the student’s interest in a boring or difficult topic.
Color diagram, like in the image below, can provide a great understanding of the process of Uracil based excision cloning. It shows the binding of one strand of DNA fragment with the vector fragment via Uracil.
Figure: An image of binding of DNA fragment with vector fragment from Labster’s virtual laboratory on Uracil based excision cloning (USER).
Before in-depth learning, students should know the basic steps of Uracil based excision cloning. It can help them to understand the Uracil based excision cloning (USER) more comprehensibly.
The first step is the identification of a DNA polymerase that can proofread the Uracil.
Vector design for Uracil based excision cloning contains an insertion of 42bp cassette.
The collection of USER vectors is formed by the conversion of established vectors.
Insertion of 13bp sequence between the fragment of DNA and the vector sequence.
Progressive Uracil based excision cloning for multiple insertions of the vector.
Additionally, the primer design for Uracil based excision cloning (USER) is divided into two types; the binding region and the 5’ overhang region. The binding region is designed by using standard rules in the standard PC. While the 5’ overhang region produces 3’ cohesive overhangs that ligate the fragments of DNA. The length of the prior overhang consists of 7 to 12 nucleotides. The ideal temperature for overhanging is 10 to 30 degrees Celsius. With the help of these rules, Uracil based excision cloning takes place and it helps to ligate the DNA fragments.
It is important to remember the functions of three enzymes involved in the Uracil based excision method. These three enzymes play key role in Uracil based excision cloning (USER).
DNA polymerase helps to amplify the uracil containing DNA fragments in the PCR. The main reason for using this enzyme in cloning is because of its thermal stability.
Another enzyme is Uracil DNA glycolysis, which helps to break the glycosidic bond between ribose and the Uracil.
Endonuclease VIII breaks the phosphodiester bond on the DNA single strand. This enzyme helps to remove the upstand DNA sequence and produce the 3’ overhangs.
Moreover, the perfect results of USER cloning depend upon the use of controls. For Uracil based excision cloning, it is necessary to have a negative control to check the efficiency of the vector.
Since Uracil based excision cloning is a complex and difficult topic, we recommend learners to use Uracil based excision cloning at Labster. At Labster, we can provide an advanced laboratory simulation that can help students to understand Uracil based excision cloning (USER) technique in an easier way.
So, check out our simulation to learn about the procedure of Uracil based excision cloning. By using this 3D stimulation, students can easily understand the principle, primer design, and molecular mechanism of Uracil based excision cloning. Students do not need to struggle anymore because labster’s gamification elements can help them in their studies. When students can see the visual lab simulation of Uracil based excision cloning (USER) technique, it will fix their mistakes. You can take advantage by using labster 3D stimulation because it can provide a piece of deep visual information about Uracil based excision cloning (USER).
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