Nucleotides are compounds composed of purine bases or pyrimidine bases, ribose or deoxyribose, and phosphoric acid. They are also called nucleotides. Five-carbon sugars and organic bases synthesize nucleosides, and nucleosides and phosphoric acid synthesize nucleotides. Four kinds of nucleotides form nucleic acids. According to different sugars, nucleotides are divided into ribonucleotides and deoxynucleotides. Depending on the base, there are adenine nucleotides (adenylic acid, AMP), guanine nucleotides (guanylic acid, GMP), cytosine nucleotides (cytidine acid, CMP), uracil nucleus Uridine acid (uridine acid, UMP), thymine nucleotide (thymidylic acid, TMP) and hypoxanthine nucleotide (inosinic acid, IMP), etc. The phosphoric acid in nucleotides has several forms, including one molecule, two molecules, and three molecules. Nucleotides are mainly involved in the construction of nucleic acids, and many single nucleotides also have a variety of important biological functions, such as adenosine triphosphate (ATP) and dehydrogenation coenzymes related to energy metabolism.
Nucleotides as substrates in kits
As a substrate for nucleic acid amplification, nucleotides are widely used in various pathogen detection kits. Nucleic acid amplification uses the target molecule as the initiating factor to synthesize the same nucleic acid copy as the target molecule. Conventional nucleic acid amplification kits include polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), and strand displacement amplification (SDA) and helicase-dependent amplification (HDA). Among them, PCR and PCR-derived technologies are the best developed and most widely used methods for nucleic acid amplification. The real-time PCR-based platform has been widely integrated into many commercial kits manufactured by Roche, Abbott Molecular, Becton Dickinson, Cepheid, Prodesse/Gen-Probe, and Eragen/Luminex. Taking the bioMeriuex Bacterial Barcode system as an example, PCR technology based on repetitive sequences has been used in clinical settings for rapid microbial strain typing and subtyping. Microbial ribosomal RNA gene amplification based on extensive PCR has been recognized as a rapid and accurate microbial pathogen identification tool. A variety of new multiplex PCR technologies have been developed and commercialized, including dual primer oligonucleotides and target-rich multiplex systems for the detection and characterization of a group of microbial pathogens.
Transcription-mediated amplification (TMA) and NASBA are involved A variety of enzymes and a series of complex reactions, they all occur simultaneously in the same temperature and the same buffer. Advantages include very fast kinetics and no need for a thermal cycler because the reaction occurs under isothermal conditions. Several TMA-based systems produced by GenProbe Inc. have been used to detect Mycobacterium tuberculosis, Chlamydia trachomatis, and Neisseria gonorrhoeae in smear-positive sputum specimens. Products based on the NASBA system have been used to detect enterovirus in cerebrospinal fluid, influenza virus, and hepatitis C virus in respiratory specimens. LAMP has been used as a major nucleic acid amplification development system to detect and identify microbial pathogens. Nucleotides are used as substrates for nucleic acid amplification in DNA synthesis and play an important role in nucleic acid detection kits, especially deoxynucleotides (dNTP).
Nucleotides as additives in kits
To ensure the accuracy of nucleic acid amplification results, non-specific PCR amplification and contamination should be prevented. A commonly used measure is to replace dTTP with dUTP in the kit, so PCR products are all DNA strands containing dU. Add a 50°C incubation step before the start of PCR. The UNG enzyme can degrade the uracil bases in the existing U-DNA contaminants in the reaction system, and under the conditions of the subsequent denaturation step, the DNA strand breaks can be eliminated. The amplification produced by contaminating DNA ensures the specificity and accuracy of the amplification results. At the same time, the UNG enzyme is inactivated and will no longer degrade the newly amplified product U-DNA.
Nucleotides as energy supply materials in kits
In addition to nucleotides playing an important role as a substrate in nucleic acid amplification, nucleotides also play a vital role in other kits, especially ATP. For example, in phosphorylation kits, phosphokinase catalyzes the transfer of phosphate groups on ATP to other compounds, and occasionally catalyzes the transfer of phosphate groups on other nucleoside triphosphates. In addition, in the ligase kit, the nucleic acid ligase uses the energy of ATP to catalyze the formation of phosphodiester bonds between two nucleotide chains. In short, nucleotides play a pivotal role as an important component in current nucleic acid commercial kits.
Reference
1. Eisenstein B I. The polymerase chain reaction: a new method of using molecular genetics for medical diagnosis[J]. New England Journal of Medicine, 1990, 322(3): 178-183.
2. Mullis K B. The unusual origin of the polymerase chain reaction[J]. Scientific American, 1990, 262(4): 56-65.
3. Ou C Y, Kwok S, Mitchell S W, et al. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells[J]. Science, 1988, 239(4837): 295-297.
4. Lalande V, Barrault L, Wadel S, et al. Evaluation of a loop-mediated isothermal amplification assay for diagnosis of Clostridium difficile infections[J]. Journal of clinical microbiology, 2011, 49(7): 2714-2716.
5. YEGUTKIN G G, HENTTINEN T, JALKANEN S. Extracellular ATP formation on vascular endothelial cells is mediated by ecto‐nucleotide kinase activities via phosphotransfer reactions[J]. The FASEB Journal, 2001, 15(1): 251-260.
6. Subramanya H S, Doherty A J, Ashford S R, et al. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7[J]. cell, 1996, 85(4): 607-615.