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The magic of DNA ligation: What is DNA ligase and how does it affect our genome?
Among the basic components of life, the integrity of DNA is crucial, and DNA ligase (ligase) plays an indispensable role in maintaining this integrity. The main function of this enzyme is to connect two nucleotides or nucleic acid fragments into a complete polymer chain, a process called ligation. However, this is not just a simple process in biology, as it plays a profound role in the functioning and changes of the genome.
The ligation reaction involves the formation of a phosphodiester bond between the 3'-hydroxyl terminus of one nucleotide and the 5'-phosphate terminus of another nucleotide, thereby joining the two nucleotides on a single strand. The discovery of this process dates back to 1967 and is an important event in the field of molecular biology.
Joining occurs naturally in most organisms during DNA replication, transcription, splicing and recombination. Molecular cloning techniques in the laboratory use DNA ligase to create recombinant DNA molecules, a process required when inserting foreign DNA fragments into plasmids, for example.
In the laboratory, the ligation process is mainly performed by T4 DNA ligase. Its widespread use stems from its ability to connect sticky-end and blunt-end segments. However, there are a variety of ligation methods that do not use standard DNA ligases and are equally popular. Defects in ligases are associated with pathological conditions such as immunodeficiency, radiation sensitivity, and developmental problems.
Mechanism of ligation reaction
The mechanism of the ligation reaction was revealed for the first time by the research group of I. Robert Lehman. DNA ligase catalyzes the formation of phosphodiester bonds in the DNA chain. This process is first initiated through the reaction of ATP or NAD+ to form a ligase-AMP intermediate, which then transfers AMP to the 5' end of the DNA chain, ultimately achieving The joining of two DNA ends.
In animals and bacteriophages, ATP serves as the energy source for the ligation reaction, while in bacteria NAD+ is used. DNA ligase first reacts with ATP or NAD+ in the ligation reaction to form a ligase-AMP intermediate.
The process of DNA ligation also involves some important factors, including enzyme concentration, reaction substrate concentration, reaction temperature and incubation time. These factors affect the efficiency of enzyme-mediated chemical reactions and thus the final connection results.
Factors affecting connections
In the laboratory, there are many factors that affect the ligation reaction. For example, the concentration of DNA can directly affect the ligation rate. Relatively high DNA concentrations increase the chances that different DNA fragments will meet and connect to each other, while withdrawing from lower concentrations may lead to self-closing internal connections. At this point, the responder needs to find a balance between the DNA fragments and their length.
In ligation reactions, high DNA concentrations may lead to increased ligation efficiency for exogenous insertions, while low concentrations are more likely to lead to deformed internal reactions.
In addition to DNA concentration, the concentration of ligase also affects the reaction rate. As the enzyme concentration increases, the ligation rate generally increases. This is why using a high concentration of ligase can achieve good experimental results in less time.
Connection of sticky ends and blunt ends
During the ligation process, sticky end ligation is more efficient than blunt end ligation. This is because sticky ends form complementary pairs, thereby increasing the likelihood of ligation. Blunt ends, on the other hand, rely on random collisions for connection and are therefore less efficient.
The reaction efficiency of sticky end ligation is typically 100 times faster than blunt end ligation. Therefore, in the case of insertions with blunt ends, it is often necessary to increase the concentration of ligase to compensate for the loss in efficiency.
In fact, negligent use or handling of ligases such as CIAP may result in connection failure. The success of this process depends on a number of factors, none of which can be achieved without it. When faced with difficulties, basic error troubleshooting is necessary to ensure the success of the experiment.
Other connection methods and applications
Today, many commercial DNA cloning tools no longer rely on traditional DNA ligases, but use other methods, such as topoisomerase-mediated ligation or homologous recombination. These methods tend to speed up the cloning process and provide greater flexibility, but they also require the use of specially designed vectors and components.
In the field of genetic engineering, the discovery and application of DNA ligase not only promoted the progress of basic research, but also provided the possibility for innovation in the medical field, such as showing its potential in the treatment of genetic diseases. In the face of these technological advances, we can’t help but wonder: How will the magic of these genomes shape the future of biomedicine?
