Introductions and Applications of Quantum Dots

Abstract: Quantum dots are semiconductor nanocrystalline materials with quantum confinement effects in three dimensions of space, also known as “artificial atoms.” The particle size of the quantum dot material is generally between 1-10 nm. Since the electrons and holes are quantum confined, the continuous band structure changes to a vertical energy level structure, thereby bringing a narrow luminescence spectrum (20-30 nm), as well as high purity, wide color gamut and other advantages. The origin of quantum dot technology dates back to the mid-1970s. It was originally developed to solve the global energy crisis. In today’s nano research field, quantum dot counting is one of the star materials. Compared with organic fluorescent materials, quantum dots have controllable emission wavelengths and research; high luminous efficiency, one-step excitation, multiple colors, strong fluorescence intensity, long-lasting anti-photobleaching, etc., which is an ideal substitute for organic fluorescent materials. Based on the above characteristics and advantages, the outstanding performance of quantum dots in life sciences and medicine has attracted wide attention. For example, hydrothermal preparation of CdSe quantum dots and their application in cell imaging, based on CdSe/ZnS quantum dots to construct dopamine fluorescence detection method and mitomycin fluorescence detection system, CdSe quantum dot synthesis and the application of heavy metal ion detection, graphene quantum dot preparation technology and the status quo of innovative resources. This paper focuses on the wide application and development prospects of quantum dots.

Keywords: quantum dots, CdSe/ZnS, fluorescence detection, development and application

The concept of quantum dots and its classification

 

Quantum dots are stable, water-soluble semiconductor nanoparticles composed of Group II-VI or Group III-V elements.

 

Quantum dots are composed of a core and a shell. The core generally uses CdX (X=S, Se, Te) as a material, and the shell portion is composed of other materials with different band gaps or a vacuum medium. Studies have shown that quantum dots can be prominent in biochemistry such as labeled peptide chains and deoxyribonucleotide long-chain polymers, which has attracted widespread attention from biologists, chemists, and physicists. Because its research direction involves many interdisciplinary subjects such as physics, chemistry, biomedicine, etc., quantum dots have considerable development and application prospects.

The application of quantum dots in the field of life science

Traditional fluorescent probes are a far cry from quantum dots as fluorescent probes. Because quantum dots have the advantages of wide laser spectrum, continuous distribution, and symmetric emission spectrum distribution, narrow width and rich color, quantum dots can solve many problems that traditional fluorescent probes cannot solve. However, the coupling problem that occurs with quantum dots of different materials is still the main problem that currently limits the development of quantum dots. Scientists predict that if the coupling problem of quantum dots of different materials is solved, the researcher can combine quantum dots as fluorescent probes with specific antibodies, and the fluorescent probes will be specific to different specific organelles in the cell. After sexual combination, different organelles are distinguished. Since a variety of semiconductor nanocrystals of different sizes can change according to the change of size, a series of marking systems with different wavelengths and different colors are produced, and the fluorescence intensity, high stability and durability make the semiconductor nanocrystals can withstand multiple times. Excited and maintained in the original state, it is convenient for the researcher’s long-term experimental observation, and the optical characteristics will not change significantly.

Fluorescence Resonance Energy Transfer (FRET) is a spectral analysis method that analyzes non-radiative energy transfer between fluorescent substances. Since its introduction, FRET has been widely used in various fields of agriculture, medicine, forensic science and scientific research. . In recent years, FRET technology has greatly improved in spatial resolution and sensitivity. Sensors designed according to FRET have been widely used in biological research fields, such as detecting conformational changes of biological macromolecules and interactions between biological macromolecules and the distance between biomolecules on the nanometer scale. Studies have shown that the optical properties of the energy donor and the assembly method of the sensor have a crucial impact on the detection performance of the FRET sensor. However, the traditional energy supply receptors (such as organic fluorescent dyes, biological materials, lanthanides, etc.) are fluorescent. Low intensity, low light stability, sensitive to the environment, poor biocompatibility, and high toxicity, easily affected by autofluorescence and stray light in the living body, resulting in the degradation of fluorescence intensity and the failure of biological detection to achieve the desired target. This limits the development and application of FRET sensors.

The FRET system in the near-infrared region has been constructed. The donor has a fluorescence emission peak at the near-infraredInP/ZnS quantum dot, and the acceptor is a near-infrared fluorescent dye Cy7. Make up for the shortcomings of traditional visible quantum dots in biological applications. At the same time, the concentration and cell microenvironment pH sensitivity of the system were tested. The results show that the fluorescence intensity of the FRET system can directly reflect the pH change of the cell microenvironment, and has high detection accuracy for the pH value of the system.

By studying the effect of different pH solutions on the FRET system, the results show that the Cy7 dye itself is not sensitive to pH. The sensitivity of the FRET system to pH is mainly due to the sensitivity of the quantum dots to pH, when the pH of the solution is at From 7 to 10 hours, the FRET system has a high FRET transfer efficiency. The cell test results show that the fluorescence signal of the FRET probe changes significantly with the change of the pH of the extracellular fluid, and can be used for detecting cancer cells in the biological microenvironment. At the same time, the obvious fluorescence signal of the FRET system in the extracellular fluid of breast cancer can be applied to the imaging of cancer cells, achieving the dual function of the FRET system.

This study provides a theoretical and experimental basis for the application of the FRET system as a sensitive probe for early diagnosis of cancer.

The application of quantum dots in medical drugs (taking CdSe and ZnS as examples)

Since quantum dots of different sizes can produce different mark colors, scientists can combine quantum dots of different sizes with different target cells to detect target cells for drug action, thereby screening drugs; it is known that centimeter-thickness of tissue, infrared rays can be Easy to penetrate and visible light can’t. Therefore, it is possible to mark the tissue with quantum dots that emit light in the infrared region, and perform medical diagnosis under the excitation of infrared light by the same detection principle as medical imaging. CdSeQDs is a kind of quantum dot which is relatively mature in research. Compared with other kinds of quantum dots, CdSeQDs has significant advantages. For example, under the same wavelength of light, the emission spectrum of CdSeQDs is 430 with different particle size. Adjustable in the range of -660nm, CdSeQDs has high fluorescence quantum yield, easy detection, mild synthesis conditions and short synthesis cycle. Therefore,CdSe quantum dots have long been widely concerned and studied.

Synthesis method of CdSeQDs

The morphology and structure of semiconductor quantum dots have a great influence on their inherent magnetic, electrical and optical properties. The properties of QDs prepared by different synthetic methods are different and their uses are different. Therefore, the synthesis of quantum dots has been subject to scientists’ wide attention. At present, organic phase synthesis and aqueous phase synthesis are two main synthetic methods. Organic phase synthesis of QDs is one of the most commonly used methods. Its development mainly includes two stages: organic metal method using organic metal as reaction precursor and green chemical method using inorganic metal as reaction precursor. The organometallic method can synthesize QDs which are monodisperse and have high stability, and the QDs prepared by this method have better fluorescence quantum yield and optical properties. However, the organometallic method also has many disadvantages, such as the use of raw materials is relatively expensive and has greater toxicity, the synthesis process is dangerous, prone to explosion, etc., which limits the further promotion and application of this method. Later, the researchers proposed a green chemical synthesis method, using high stability, low toxicity and low cost cadmium oxide as raw material instead of dimethyl cadmium as a precursor, mixed with Se solution to prepare CdSeQDs with excellent properties. Although this method reduces the reaction cost, the use of a more toxic organic phosphine solvent is retained during the reaction, so a greener synthesis method needs to be sought. The preparation of QDs by aqueous phase method overcomes the shortcomings of organic synthesis, but the method also has problems such as imperfect crystals, many surface defects and poor luminescence properties, so the aqueous phase synthesis method needs further improvement.

Surface functionalization of CdSeQDs

Although CdSeQDs have many unique and excellent physicochemical properties and optical properties, they also have some limitations, such as low biocompatibility, poor water solubility, and relatively high toxicity. In order to overcome these shortcomings, the surface of quantum dots needs to be encapsulated and modified. In recent years, various small molecules (including sulfur, polymer, DNA and other modifiers) have been reported to encapsulate modified CdSeQDs. The prepared QDs have good biocompatibility and are widely used in the detection of biological small molecules, disease diagnosis and treatment, biosensing, etc., and have broad application prospects in the field of biomedicine.

Protein package

Protein-encapsulated QDs have many important applications in the biomedical field, so proteins are used as a common encapsulant for the synthesis of QDs. Proteins can usually be modified to the surface of QDs by electrostatic attraction, covalent bond coupling, etc., where covalent bonding is the most common method. Covalent bond bonding is a combination of a functional group of a protein molecule and a functional group on the surface of a quantum dot to form a covalent bond. Since the protein has an amino group and a sulfhydryl group, it is easy to form a covalent bond with the amino group and the carboxyl group-modified QDs. The use of novel proteins (natural phytochelatins) bound to poorly water-soluble CdSe/ZnSQDs increases the water solubility of QDs, and the encapsulated QDs exhibit high colloidal stability and remain in aqueous solution, including its high quantum yield, capable of bioconjugation with different functional groups.

The following figure is a schematic diagram of fluorescence resonance energy transfer caused by self-assembly of GCN-mCherry and glutathione-coated CdSe/ZnS quantum dots.

Antibody package

An antibody is a glycoprotein capable of specifically binding an antigen. Attaching an antibody to a quantum dot enables specific binding of an antibody on the QDs to an antigen in the living body, thereby enabling qualitative and quantitative detection of biomolecules in the living body. When a disease occurs in an organ in a living body, it causes overexpression or decreased expression of certain molecules in the organ; therefore, antibody-modified QDs can detect tumor cells as fluorescence sensors in vivo. The binding method of antibody and quantum dot is divided into non-covalent binding and covalent binding, and non-covalent binding mostly adopts biotin-avidin linkage. Covalently bound to the surface functional group of the antibody and the quantum dot reacts with each other by the activation of dimethylaminopropyl and ethylcarbodiimide hydrochloride to form a covalent bond. The most commonly used combination is amino and carboxyl. The most commonly used combination is an amino group and a carboxyl group, an amino group and a thiol group, and an aldehyde group and a hydrazide.




 

Peptide wrap and DNA wrap

Peptides are often used to modify QDs as common biomolecules. The combination of peptides and quantum dots often utilizes the amino and sulfhydryl groups on the surface of the peptide to covalently bond with the amino and carboxyl groups on the surface of the quantum dot. In addition, the method of synthesizing small molecule-encapsulated QDs by biomimetic method is relatively simple and has been widely promoted. Usually, phospholipids are also used to wrap QDs.

DNA-functionalized QDs play an important role in biological applications. Single-stranded DNA recognizes complementary DNA/RNAs that form a normal helical structure through base-pair pairing, so DNA-functionalized QDs can detect viruses and bacteria. DNA fragments as well as small RNAs, DNA can also act as aptamers with high affinity to target molecules. These aptamer-functionalized quantum dots can be used to detect ions, proteins, or for disease diagnosis and cell imaging.

Construction of mitomycin fluorescence detection system based on CdSe/ZnS quantum dots

In the development of the past few decades, experts have studied the optimal reaction conditions of quantum dots in detail, and developed a new method for detecting mitomycin in biological fluid based on the fluorescence properties of CdSe/ZnS quantum dots. The intensity has a good linear relationship with the concentration of mitomycin. Under the optimal experimental conditions, the CdSe/ZnS quantum dots interacted with mitomycin, and the fluorescence intensity of the quantum dots was quenched with the increase of mitomycin concentration. The quenching mechanism causes photo-induced electron transfer to cause quantum dot fluorescence quenching. The detection method is simple in operation, high in sensitivity, and has good applicability to analysis of actual samples. In addition, based on CdSe/ZnS quantum dots, the study of dopamine fluorescence detection methods can be constructed, and corresponding results have been obtained.

 

Application of quantum dots in fingerprint visualization

In 2000, MENZEL in the United States reported for the first time the use of CdS quantum dots for the fingerprinting of cans on the surface of cans, creating a precedent for the application of quantum dots as a new material in fingerprint visualization. Subsequently, MENZEL et al. used PAMAM (polyamide) as a template to control the growth of CdS quantum dots encapsulated in dendrimers by the spatial threshold effect of dendrimers. The synthesized CdS/PAMAM polymer was diluted with methanol as solvent. After that, it was successfully used for the appearance of latent fingerprints on aluminum foil and polyethylene samples. They believe that functional groups such as amino or carboxyl groups on the surface of CdS/PAMAM can interact with fingerprint residues to deposit CdS/PAMAM onto the fingerprint lines, and the fingerprints appear through the fluorescence of CdS/PAMAM polymer under ultraviolet light. The method can be used for the identification of surface fingerprints of various objects (such as transparent tape, black plastic bags, tin foil, etc.) of different background colors.

The above is a comparison of fingerprint effects with CdS/PAMAM polymer and traditional visualization reagents. As you can see, the fingerprints displayed with CdS/PAMAM polymers are clearer(The picture on the left is CdS/PAMAM).

Application of quantum dots in improving solar cells

Solar cells, as the name implies, are devices that make full use of the energy of sunlight and convert solar energy into electrical energy through the photoelectric effect. Ordinary solar cells cannot fully utilize solar energy, and heat loss during conversion is large, resulting in defects in low photoelectric efficiency conversion rate. Scientists have used silicon semiconductor materials as a medium for solar and electrical energy conversion to develop common semiconductor materials for solar cells. After the silicon semiconductor material is irradiated by the sun, the internal electrons move freely to form a current. It is this special property of semiconductor materials that semiconductor solar cells can operate normally, but the results in actual production are not satisfactory. Scientists have learned that currents are generated only when these free electrons are moved to the electrodes. In fact, there are few free electrons moving to the electrodes, resulting in low conversion to solar energy. Therefore, ordinary semiconductor materials cannot completely convert solar energy into electrical energy.

Scientists envisaged that if the theory of quantum mechanics was introduced into the solar cells of ordinary semiconductor materials, it would be able to make up for the shortcomings of the actual power generation efficiency of ordinary semiconductor materials. Scientists conducted a large number of theoretical calculations, and the results were quite shocking to the people present. This kind of battery material is not only cheaper, but also can make full use of the sunlight energy while the consumption in the conversion process is minimal, so that the conversion rate of the solar cell to the photoelectric is doubled. Although the current super battery is still under development, whether it is as good as scientists expected and when it will be released to the public is still unknown. However, it is undeniable that quantum dot solar cells are becoming a new trend in research and development and are currently the most concerned research topic. It is believed that this super battery can completely solve the shortcomings of high energy consumption and low photoelectric conversion efficiency of ordinary semiconductor materials. Quantum dot solar cells will show excellent skills and bright prospects in future solar energy conversion and utilization.

Quantum dot sensitized solar cells are one of the quantum dot solar cells that have been developed so far. A quantum dot sensitized battery is a battery between a common semiconductor material solar cell and a super cell (quantum dot solar cell). So quantum dot sensitized cells cannot be called true quantum dot solar cells, it is just a transitional product. Quantum point solar cells in the true sense are still in the research and development stage. Quantum dot solar cells need to be studied in the way of photoelectric conversion, and the selection of medium is also necessary. The medium is the focus and hot spot of research on super batteries. The dielectric materials of super batteries must have ultra-high conversion rate and low cost, quantum dots. There is still a long way to go before solar energy research and development. As a transition product, quantum dot sensitized solar cells are based on dye-sensitized solar cells (DSSC) and are designed and developed using similar working principles. The quantum dot sensitized solar cell photoanode is composed of a quantum dot attachment and a photogenerated electron injection carrier, and the carrier is mainly composed of a binary semiconductor oxide.

The development prospect of quantum dots

As a new research field, quantum dots have attracted the attention of scientists in many fields in a short time. Scientists are actively involved in the study of quantum dots, which further demonstrates that the study of quantum dots is related to the advancement of science and technology and the development of science. Quantum dot technology is still in the early stage of theory, and scientists have found that quantum dots play an important role in life sciences and medicine. Scientists envision that if we can fully apply quantum dots to our lives, our lives will be more convenient and better. This is a perfect flaw for scientists and everyone. Scientists are fully devoted to the study of quantum dots, trying to put the application of quantum dots from theory to life and put them into practice. Quantum dot technology is a high-tech. Scientists have discovered the outstanding performance of quantum dots as fluorescent probes, the application of quantum dots in medical drugs, and the application of quantum dots in improving solar cells. Quantum dots are A little bit infiltrated into our lives. The research of quantum dots is still in its infancy. With the advancement of society and the continuous development of science and technology, the research scope of quantum dots is gradually expanding. As a new concept, quantum dots are well known. There are more mysteries in semiconductor microcrystals waiting for us to explore and discover. The study of quantum dots is moving at a strong pace, and the application of quantum dots is showing bright prospects.

Functional Overexpression and Purification of Membrane Transport Protein

One of the basic characteristics of living cells is the ability to exchange substances and information with the external environment. Through the physiological activities of material and information exchange, cells can sense environmental changes, acquire nutrients necessary for metabolism, and discharge metabolites and waste. The selective absorption and discharge of solutes is mainly achieved by a membrane transport protein-based transport system on the cell membrane.

What are transport proteins?

Transport proteins are a large class of membrane proteins that mediate chemicals and signals exchange inside and outside biofilms. The lipid bilayer forms a hydrophobic barrier around the cell or organelle that isolates it from the surrounding environment. Although some small molecules can penetrate directly through the membrane, most hydrophilic compounds (such as sugars, amino acids, ions, drugs, etc.) require the help of specific transporters to pass through the hydrophobic barrier. Therefore, transporters play an important role in a wide range of cellular activities such as nutrient uptake, release of metabolites, and signal transduction.

The isolation and purification of proteins are the basis for the study of the structure and function of proteins. Under normal circumstances, the expression of most membrane proteins is extremely low, and the bottleneck of membrane protein structure and function research is the lack of effective expression of membrane proteins. Purification techniques are effective means of conducting structural studies. Compared with other types of membrane proteins, membrane transporters are basically single-gene coding products, which can independently perform physiological functions and become a good functional expression and purification research object. The currently used purification methods are: (1) using molecular biology techniques to construct a recombinant membrane protein containing a fusion affinity tag; (2) optimizing the functional expression of the recombinant membrane protein; (3) and isolating and purifying the expressed Membrane protein, and activity testing and structural studies. The advantage of this strategy is that, on the one hand, the introduction of the fusion affinity tag facilitates the detection and purification of the recombinant membrane protein; on the other hand, it is easy to modify and manipulate the target protein, and these modifications and operations such as site-directed mutagenesis can provides important information for the study of protein structure and function.

Construction of recombinant membrane transport protein

When constructing a recombinant membrane protein for in vivo expression, the topological structure of the protein must be considered, especially whether the N-terminus and C-terminus of the expressed membrane protein are located on the cytoplasmic side or the outside of the cell. On the one side, heterologous overexpression of membrane transporter is affected by the N-terminal sequence of the first transmembrane helix located outside the lipid membrane. When constructing a recombinant membrane transporter, whether to introduce a signal sequence-promoting protein based on the position of the N-terminus of the membrane protein expression. In the construction of its expression vector, especially when it is necessary to introduce an affinity tag at the N-terminus, it is necessary to simultaneously introduce and contain a signal sequence in order to successfully express the target protein. Other side, depending on the topological structure of the membrane transporter and the polarity of the amino acid side chain, the type and position of the affinity tag to be introduced can be selected. The affinity tag is an amino acid polypeptide or protein, and an affinity tag is introduced into the recombinant protein. The purpose is to determine whether the target protein is expressed and affinity-purified the target protein. The common feature of the affinity tag is that it can bind to a certain affinity medium, thereby facilitating the affinity purification of the recombinant protein containing the affinity tag. Affinity labeling needs to follow the principle of positively charged amino acid inward, otherwise the expression of the target protein containing the affinity tag will be affected.

Functional overexpression of recombinant membrane proteins

According to the topological properties of membrane proteins, we need to select appropriate expression strategies and optimize expression conditions to obtain a large number of active membrane proteins, which facilitates subsequent purification and structural function studies. Factors that limit membrane protein expression include: lacking of efficient membrane protein folding mechanism or stabilization mechanism in the expression host; degradation of protease; toxicity of recombinant membrane protein to host; inefficiency of protein translation caused by codon preference; post-translational processing of protein Modified or missing modifiers.

Depending on the source of the membrane protein expressed, alternative efficient expression systems include living expression systems such as prokaryotic expression systems and eukaryotic expression systems; and expression systems in vivo. For membrane proteins derived from prokaryotes, the E. coli expression system has achieved good results. The expression and folding of most membrane proteins of Gram-positive bacteria such as E.coli are related to signal recognition particles (SRP). At the same time, inserting into the membrane under the action of SRP completes the folding. The optimization of the functional expression of the recombinant membrane protein aims to control the relative speed of translation and folding to achieve the optimal expression of membrane protein. SRP-mediated membrane protein folding. It also relates to the recognition and folding efficiency of heterologous recombinant membrane protein by the host cell folding mechanism. Therefore, it is necessary to optimize the factors affecting membrane protein translation and folding, for example, the homology of the expression host and heterologous membrane protein source, culture temperature, the composition of the medium, and the choice of co-expressed protein, etc.

Functional expression of membrane proteins derived from eukaryotes often involves a series of processes in protein processing and sorting, and prokaryotic expression systems often fail to achieve satisfactory results. The use of weak protein promoters and the simultaneous expression of molecular chaperones can increase the efficiency of ion channel protein expression. These studies demonstrate that membrane proteins require a certain amount of time and appropriate mechanisms for processing and folding, and ultimately localization, after being synthesized by ribosomes. This is important for the functional expression of membrane proteins. In the living expression system, many factors can lead to the inefficiency of membrane protein expression, for example, the expressed membrane transporter has toxic effects on host cells. In this case, the expression of membrane transporter can be considered by using the in vitro expression system.

In vitro expression systems can also cost-effectively label target proteins for specific studies. The membrane proteins expressed in vitro exist in agglomerated state. After the detergent is added, most of the membrane proteins can be dissolved. The CD structure shows that the secondary structure is mainly α-helix, and the dissolved EmrE, SugE and TehA can be recombined. On the artificial lipid membrane. The transport activity of EmrE recombinant membrane proteoliposome showed that the in vitro expressed EmrE has transport activity. The detergent has two polarities and is stable to the stability of the membrane protein in the membrane-free state. If a suitable detergent is directly added to the in vitro expression system, a membrane transporter present in a dissolved state can be obtained. These membrane proteins can be directly used to construct recombinant membrane proteoliposomes. Studies on MscL recombinant membrane proteoliposomes showed that MscL expressed in vitro after addition of detergent has similar activity to MscL expressed in vitro. Therefore, the membrane transports protein with suitable conformation and functional activity can be obtained by using the in vitro expression system.

Selection of detergents and purification of recombinant membrane proteins

Purification of recombinant membrane proteins containing fusion affinity tags involves three major steps: releasing of membrane proteins from the plasma membrane with a suitable detergent; adsorption of free membrane proteins with affinity chromatography or affinity media; washing away impurities and elution membrane protein. The choice of detergent in this process must consider the following two points: (1) the solubilizing efficiency of the detergent on the target membrane protein; (2) how to remove the detergent after purification for structural and functional studies. Membrane proteins require a plasma membrane to support their structure and function in their natural state, but how to maintain the stability of membrane protein structure and function under the condition of membrane removal during separation and purification is important. Usually, the detergent interacts with the membrane protein and the hydrophobic part of the membrane lipid to weaken the hydrophobic binding between the membrane protein and the membrane lipid molecule, and release the membrane protein from the membrane lipid. The hydrophobic portion of the free membrane protein retains its original conformation under the protection of the detergent, thereby maintaining its original function, such as binding to substrates or inhibitors. Detergents are divided into ionic and nonionic types. Non-ionic detergents and amphoteric detergents have a weaker denaturation effect on membrane proteins, and are commonly used for ion exchange and affinity chromatography of membrane proteins. The solubilizing efficiency of the detergent on the membrane protein is related to the structure of the detergent and the critical micelle concentration (CMC), which can be optimized by changing the type and concentration of the detergent. The appropriate detergent will contain the affinity label. After the recombinant membrane protein is dissolved from the plasma membrane, the affinity membrane is adsorbed by the affinity chromatography column or the affinity medium to carry out the affinity purification experiment. The purification step is similar to the affinity purification step of the soluble protein. However, it must be noted that the concentration of the detergent in the solution must be kept above the critical micelle concentration, while the solution is required to contain high concentrations of glycerol (20%) to maintain the hydrophobic conformation and functional stability of the membrane protein.

In the state of membrane removal, even with the synergistic effect of detergent and glycerin, the stability of most membrane proteins is not strong. Therefore, after purification, membrane proteins must be recombined into artificial lipid membranes as needed to construct membrane protein liposomes, and restore the structure and function of membrane proteins on lipid membranes. In this process, the concentration of detergent should be reduced below its critical micelle concentration, so that the membrane protein is detached from the detergent and inserted into the lipid membrane to form artificial membrane proteoliposome. Methods for removing the scale or reducing the concentration of the detergent include a dilution method, a gel filtration method, a dialysis method, an ion exchange chromatography method, and a special medium absorption method. When using gel filtration and ion exchange chromatography, it is not conducive to the membrane protein interaction with membrane lipid molecules while removing detergent. Dialysis removes detergents at a slower rate and is commonly used for two-dimensional crystallization experiments of membrane proteins. When constructing artificial membrane proteoliposome for functional activity studies, descaling is usually removed by dilution or Biobeads absorption. The use of detergents with high critical micelle concentration facilitates the rapid removal of detergents from the system, so the detergent is exchanged while the membrane protein is affinity purified, and the detergent with low critical micelle concentration is exchanged high. Critical micelle concentration of detergent.

A Detailed Introduction for Oligopeptide Synthesis

A classification of polypeptides, generally having a molecular weight range of less than 1000 Daltons, also known as small peptides, oligopeptides, oligopeptides, or small molecule active peptides, generally composed of 2- to 6 amino acids, more than A polypeptide called a polypeptide and having more than 50 amino acids is called a protein. The difference with other peptides is that they can be directly absorbed in the human body without digestion. Oligopeptides can be further divided into: oligopeptide-1, oligopeptide-3, oligopeptide-5 and the like, and oligopeptide-6 is also called hexapeptide or hexapeptide.

 

Absorption theory

The traditional theory of protein digestion and absorption believes that the protein is in the intestinal lumen and is formed by peptide protease and chymotrypsin to form free amino acids and oligopeptides (containing 2 to 6 amino acid residues). The oligopeptide is completely hydrolyzed by peptidase. It becomes a free amino acid and enters the blood circulation as a free amino acid. According to this theory, protein provides amino acids only to the animal’s body, that is, the nutrition of the protein is the nutrition of the amino acid. Therefore, as long as we provide the animals with sufficient essential amino acids, the animals will be able to obtain satisfactory performance. However, many studies have shown that the amount of proproteins that can be replaced by monomeric amino acids is limited. Feeding low levels of protein to livestock and supplementing synthetic amino acid diets does not yield optimal performance and feed efficiency. To achieve both of these goals, the diet must have the lowest amount of proprotein and oligopeptide.

 

Transport mechanismThe intact peptide enters the epithelial cells, and the presence of absorption pathways within the cell is neglected for a considerable period of time. The possibility of peptide transport was mentioned more than 100 years ago (Matthews, 1987). Agar (1953) confirmed the transport of intact di-glycans across the intestinal epithelium of rats. However, due to the influence of traditional protein digestion and absorption theory, scholars are not easy to accept other absorption methods, and because the diglyceride is considered to be a special dipeptide, its molecular weight is small, so the importance of this discovery. Not recognized. It was not until the 1960s that Newey and Smyth (1959, 1960) provided information on the complete absorption of peptides for the first time. They found out. The digestion products of proteins in the small intestine not only have amino acids, but also a large number of oligopeptides, and the peptides can enter the intestinal mucosal cells intact and further hydrolyze in the mucosal cells to form amino acids into the blood circulation.

 

Later, between 1965 and 1980, more and more evidence was accumulated about the intestinal transport of intact short peptides. In particular, David M. Matthews of London and Siamak A of Pittsburgh, Adibi’s team confirmed that the possibility of peptide transport is not only academically meaningful, but may represent an absorption pathway for amino acid nitrogen uptake that is as important as the corresponding free amino acid uptake. . The absorption process of amino acids and peptides by the intestinal mucosa is complicated. It is generally believed that dipeptides and tripeptides are absorbed into intestinal cells and then hydrolyzed by peptidases into the blood circulation in the form of free amino acids. Nutritional physiology and pharmacological tests have confirmed that in some cases intact peptides can enter the circulation through the peptide carrier of the intestinal mucosa.

 

Although the transport mechanism of oligopeptides is not fully understood, three can be confirmed: 1. The pH-dependent H+/Na+ exchange transport system does not consume adenosine triphosphate (ATP); 2. The active process depends on the concentration of H+ or Ca2+. , to consume ATP; 3, glutathione (GSH) operating system. Although the mechanism of animal oligopeptide transport is not fully understood, the transport of oligopeptides requires the recognition of vectors, and some mammalian small peptide vector genes have been cloned. By studying the structure and function of oligopeptide carriers, revealing the relationship between the carrier and oligopeptides and related ions, it is a hotspot in the research of oligopeptide transport mechanism. Many new scientific and technological achievements, such as enzymatic peptides, have been obtained in this respect. Bioactive peptides are based on food protein macromolecules containing high-quality protein, and are spliced ??and modified into the most active oligopeptides between macromolecular proteins and amino acids by using enzymes (enzyme scissors). Molecular active polypeptide, which has the characteristics of small molecular weight, easy absorption, and high nutritional value.

 

Absorption characteristics1. Do not need to digest, directly absorbed.2. No need to consume human energy when absorbing.3. Will not increase the burden of human gastrointestinal function.4. It has the characteristics of preferential absorption.5. Promote the body’s absorption with its own energy.6. Force the body to absorb when the body’s absorption function is lost.7. 100% is absorbed by the body.8. Fast absorption. It is 129,600 seconds faster than the body’s absorption of macromolecular proteins and 64,800 seconds faster than human amino acids.9. With a carrier role. Other nutrients for human consumption can be carried on the body and transported to human cells and tissues.10. Become a means of transportation in the human body and transport various trace elements to various parts of the human body.11. Has a strong diversity of activities and physiological functions.

 

Application fieldAccording to experts, oligopeptides with low molecular weight can have higher skin permeability than peptides, and are more easily absorbed by human skin. At the same time, due to the small molecular weight, biological activity has a qualitative leap. The smaller the molecular weight of the peptide, the shorter the “amino acid chain” and the easier it is to be absorbed and utilized by the body.

Expert analysis, due to OCO’s unique biological personality and outstanding functional performance, it has a huge application space in the field of daily cosmetics, shampoo and hair care, food and health products, biomedicine, and even textiles. Oligopeptides are widely used in the beauty field, leading the new trend of healthy skin care in China’s high-end beauty market.

 

About us

Creative Peptides is specialized in the process development and the manufacturing of bioactive peptides. We are dedicated to offering custom peptide synthesis, process development, GMP manufacturing as well as catalog products for customers in industry and research area.here are some our products like: Peptide Modification,De novo Peptide Design,Long Peptides Synthesis,Peptide Drug Discovery,etc.

Several Structural Functions of Viral Genome

The virus is the simplest organism. The complete virus particles include the coat protein and the internal genomic DNA or RNA (some coat proteins outside the coat have an envelope composed of host cells containing the glycoprotein encoded by the viral gene. The virus cannot replicate independently, it must enter the host cell to make the virus replicate by means of some enzymes and organelles in the cell. The function of the coat protein (or envelope) is to recognize and invade specific host cells and protect the viral genome from nucleases Destruction.

Structural features

  1. The size of the virus genomeis quite different. Compared with bacteria or eukaryotic cells, the genome of the virus is very small, but the genomes of different viruses are also very different. For example, hepatitis B virus DNA is only 3 kb in size and contains less information. It can only encode 4 proteins. The genome of poxvirus is 300 kb, which can encode hundreds of proteins, not only for the enzymes involved in viral replication. Even the enzymes for nucleotide metabolism are encoded, so the poxvirus is much less dependent on the host than the hepatitis B virus.
  2. The viral genome may consist of DNA or RNA. Each virus particle contains only one nucleic acid, or DNA or RNA, and the two generally do not coexist in the same virus particle. The DNA and RNA constituting the viral genome may be single-stranded or double-stranded, and may be a closed-loop molecule or a linear molecule. For example, papillomavirus is a closed-loop double-stranded DNA virus, while the genome of adenovirus is linear double-stranded DNA, poliovirus is a single-stranded RNA virus, and the genome of reovirus is double-stranded. RNA molecule. In general, most DNA viruses have genomic double-stranded DNA molecules, while most RNA viruses have single-stranded RNA molecules.
  3. The genome of most RNA viruses is composed of continuous ribonucleic acid strands, but some genomic RNAs of viruses are composed of discrete nucleic acid strands. The genomic RNA molecules of influenza viruses are segmental and consist of eight RNA molecules. Each RNA molecule contains information encoding a protein molecule; the reovirus genome consists of a double-stranded segmental RNA molecule with a total of 10 double-stranded RNA fragments, each of which encodes a protein. At present, no viral genome composed of segmental DNA molecules has been found.
  4. Gene overlap means that the same DNA fragment can encode two or even three protein molecules. This phenomenon is only found in mitochondria and plasmid DNA in other biological cells, so it can also be considered as a structural feature of the viral genome. This structure enables smaller genomes to carry more genetic information. The overlapping genes were discovered by Sanger in 1977 when studying ΦX174. ΦX174 is a single-stranded DNA virus, the host is Escherichia coli, and therefore, it is a phage. It infects E. coli and synthesizes 11 protein molecules with a total molecular weight of about 250,000, which is equivalent to the amount of information contained in 6078 nucleotides. The viral DNA itself has only 5375 nucleotides, which can encode a protein molecule with a total molecular weight of 200,000. Sanger can’t solve this contradiction for a long time before clarifying that some of the 11 genes of ΦX174 overlap. There are several cases of overlapping genes:

(1) One gene is completely inside another gene. For example, genes A and B are two different genes, and B is contained in gene A. Similarly, gene E is within gene D.

(2) Partial overlap. For example, gene K overlaps with a part of genes A and C.

(3) Only one base overlap of the two genes. For example, the last base of the stop codon of gene D is the first base of the J gene start codon (such as TAATG). Although these overlapping genes are mostly identical in their DNA, the protein molecules produced are often different because the reading frame is different when the mRNA is translated into a protein. Some overlapping genes have the same reading frame, but the starting sites are different. For example, in the SV40 DNA genome, there are 122 base overlaps between the three coat proteins VP1, VP2 and VP3, but the codons are not identical. While the small t antigens are completely within the large T antigen gene, they have a common start codon.

  1. Most of the viral genome is used to encode proteins, only a very small one is not translated, which is different from the redundancy of eukaryotic DNA. For example, the part that is not translated in ΦX174 only accounts for 217/5375. G4DNA accounts for 282/5577, less than 5%. The untranslated DNA sequence is usually a control sequence for gene expression. For example, the sequence between the H gene and the A gene of ΦX174 (3906-3973), a total of 67 bases, includes a control region for gene expression such as an RNA polymerase binding site, a transcription termination signal, and a ribosome binding site. Papillomavirus is a type of virus that infects humans and animals. The genome is about 8.0Kb, and the untranslated part is about 1.0kb. This region is also a regulatory region for other gene expression.
  2. Genes of functionally related proteins or genes of rRNA in viral genomic DNA sequences tend to cluster at one or several specific sites in the genome to form a functional unit or transcription unit. They can be transcribed together into a molecule containing multiple mRNAs, called polycistronie mRNA, which is then processed into template mRNA for various proteins. For example, the late gene encoding the adenovirus encodes 12 coat proteins of the virus. When the late gene is transcribed, it generates a polycistronic mRNA under the action of a promoter, and then processes it into various mRNAs, which encode various coat proteins of the virus. Functionally related; the DEJFGH gene in the ΦX174 genome is also transcribed in the same mRNA and then translated into various proteins, of which J, F, G and H are all coding for coat proteins, assembly of D proteins and viruses. Relatedly, the E protein is responsible for the lysis of bacteria, which are also functionally related.
  3. Except for retroviruses, all viral genomes are haploid, and each gene appears only once in the viral particle. There are two copies of the retroviral genome.
  4. The gene of bacteriophage (bacterial virus) is continuous; while the gene of eukaryotic virus is discontinuous, with introns, except for positive-strand RNA viruses, the genes of eukaryotic viruses are first transcribed into mRNA. The precursor is processed to remove the intron into mature mRNA. More interestingly, some eukaryotic introns or parts of them are introns for one gene and exons for another. This is the case with early genes such as SV40 and polyomavirus. The early genes of SV40, namely the big T and small t antigen genes, were all counterclockwise from 5146, the large T antigen gene was terminated to 2676, and the small t antigen was terminated to 4624, but from 4900 to 4555. A 346 bp fragment is an intron of the large T antigen gene, and the DNA sequence from 4900-4624 in the intron is a small t antigen encoding gene. Similarly, in polyomaviruses, the introns in the large T antigen gene are the genes encoding the T and t antigens.

Bovine papillomavirus genome structure and function

Papillomavirus is a DNA virus that infects human and animal skin, mucous membranes and causes papilloma lesions. It belongs to the papovavirus family. It can be divided into bovine papillomavirus (BPV), human papillomavirus (HPV), etc. depending on the host infected with the virus. The papillomavirus genomes that have been discovered so far have similar structures. The genomic structure and function of papillomavirus are illustrated by BPV as an example. The BPV DNA is 7945 bp in length and is a closed-loop supercoiled structure, which can bind to histones to form nucleosomes in host cells. The first base G of the single HpaI restriction site in the BPV DNA is the 1st position, and the base number is located in the direction of 5’→3′. DNA sequence analysis showed that all open reading frames (ORFs) existed on one DNA strand, and the genes overlap each other. The entire BPV gene component is a coding region and a non-coding region (NCR), and the coding region is divided into an early transcriptional functional region (E region) and a late transcription functional region (L region) according to the function of the encoded protein. 1. The non-coding region (NCR) non-coding region, also known as the upstream regulatory region (URR) or the long control region (LCR), is located between the late gene L1 stop codon and the first gene E6 first start codon, and the length is Different in different papilloma viruses, it is about 1.0 kb in BPV. In the promoter sequence of NCR transcription, transcription and expression of early genes can be initiated. In addition, there are enhancer sequences in this region, which can be activated by the early gene product E2 protein, further promoting the expression of early gene AAC, which has been clarified. The sequence of the enhancer of the BPVNCR region, which is a palindrome of TTGGCGGNNG and ATCGGTGCACCGAT. It can be seen from the structural characteristics of NCR that its main function is to regulate the expression of BPV gene.

  1. Early transcriptional functional region (or early gene region, E region) The E region of BPV contains eight open reading frames (ORFs), namely E6, E7, E8, E1, E2, E3, E4, E5, of which E6, E7 and E1 genes partially overlap, E8 is completely in E1, E3 and E4 are all contained in E2, and E5 and E2 partially overlap. The protein product encoded by the E2ORF can bind to the enhancer of NCR to increase or decrease the expression level of the early gene.

In addition, the E2ORF synergizes with the E1ORF to maintain the free state of the papillomavirus DNA without integration into the host cell chromosome. The proteins encoded by E6 and E7ORFs may be oncogenic proteins. E6 and E7 proteins can cause malignant transformation of the host into tumor cells. The mechanism of cell transformation induced by E6 and E7 proteins is not clear at this stage, but there are two explanations. [1] The Cys-xx-Cys repeat sequence was found in the amino acid sequence of the E6 and E7 proteins, and the structure is considered to be a specific structure possessed by the intracellular nucleic acid binding protein, and thus the E6 and E7 proteins are considered to be DNA-binding proteins. It can regulate the activity of genes, further affect the proliferation and differentiation of host cells, and make the process uncontrolled to form tumors. [2] Recently, two proteins with molecular weights of 53KD and 106KD were found in normal cells, respectively, called p53 and p106. protein. The loss or inactivation of these two proteins often causes cell malignancy. The study found that the E7 and E6 proteins of papillomavirus can be inactivated by binding to p53 and p106 proteins, respectively, which may also be a mechanism by which E6 and E7 proteins cause cell malignancy.

  1. Late transcriptional domain (late gene region, L region): There are two L-region ORFs, L1 and L2 ORF, which encode the coat protein of papillomavirus, in which L1 protein is the major coat protein and L2 protein is the minor coat protein.

Genomic structure and function of RNA phage

The most well-studied E. coli RNA phage are MS2, R17, f2 and Qβ. Their genomes are small, ranging from 3,600 to 4,200 nucleotides, and contain four genes. MS2.R17 and f2 have almost the same genomic structure. Two of the four genes encode structural proteins of phage: one is the gene of protein A, which is 1178 nucleotides in length. The function of protein A (called a mature protein) is to enable the phage to recognize the host and allow its RNA genome to enter the host bacterium, and each phage typically has only the protein A of the molecule. Another structural protein gene is 399 nucleotides in length and encodes a coat protein to form a viral particle, each of which has 180 molecules. The rest of the genome encodes an RNA replicase and a lytic protein. The gene encoding the lytic protein partially overlaps the coat protein and the replicase gene, but the reading frame is different from the reading frame of the coat protein. There are many secondary structures in the MS2, R17, and f2 genomes, and self-pairing of bases in RNA molecules may have a role in preventing RNase degradation. In addition, there is a non-translated sequence at the 5′ and 3′ ends of the coding gene, which also has a role in stabilizing RNA molecules. The genome of another RNA phage Qβ is slightly larger than the genome of the above RNA phage; [1] there is no independent lytic protein gene, but the structural protein A2 (or mature protein, Maturation Protein) has the function of dissolving protein. [2] also encodes another coat protein A1.

Structural features and functions of the hepatitis B virus genome

The genomic DNA structure of hepatitis B virus (HBV) is very peculiar and is a circular partial double helix structure with a length of about 3.2 kb. Two-thirds of them are double-helix and 1/3 are single-chain, which means that the two chains in DNA are not equal in length. The 5′ end of the long chain is not covalently linked to the 3′ end, but is covalently linked to a protein. The 5′ end of the long chain is complementary to 250-300 base pairs. The long chain is a negative chain and the short chain is a positive chain. The length of the short chain varies from virus to virus and is generally about 1.6-2.8 kb long, about 2/3 of the long chain. The gap between the short strands can be filled by a DNA polymerase in the viral particle. Hepatitis B virus is currently known as the smallest double-stranded DNA virus that infects humans. In order to replicate independently in cells, the virus contains as much genetic information as possible in a small genome. Therefore, the genomic structure of HBV appears to be particularly precise and concentrated, making full use of its genetic material.

There are many overlapping gene sequences, and there are four open reading frames in the HBV genome, which encode the nucleocapsid (C) and envelope (S) proteins of the virus, viral replicase (polymerase) and a seemingly virus Gene expression related to protein X. The two small ORFs in front of the S gene belong to the same reading frame as the S gene ORF. The ORFS can be read through and encode two S protein-associated antigens. These two antigens are also present on the surface of the virus particles. They are called pre-S1 (pre-S1) and pre-S2 (pre-S2), respectively. Similarly, there is a short ORF in front of theORF, called pre-C (pre-C), which encodes a larger C-protein associated antigen. All of these ORFs are on the negative strand DNA (long chain), in which the S gene is completely overlapped with the polymerase gene, the X gene overlaps with the polymerase gene and the C gene, and the C gene overlaps with the polymerase. Recently, Miller et al. found two ORFs, ORF-5 and ORF-6, in the HBV genome. These two ORFs overlap with the X gene, and ORF6 is not encoded by negative strand DNA, but is encoded by positive strand DNA. The function of these two ORFs is currently unclear.

The regulatory sequence is located inside the gene, which is also a way for HBV to save on the use of genetic material. Sequences involved in HBV group replication are: short-chain forward replication sequences (DR1 and DR2) and U5-like sequences (named for similar faces to the U5 sequence at the end of the retrovirus). DR1 and U5 are located in the pre-CORF and are the starting site for the long chain of synthetic DNA. DR2 is located at the overlap of the polymerase gene and the X gene and is the starting site for DNA short-chain synthesis.

There are four signal sequences involved in HBV gene expression: [1] promoter, [2] enhancer, [3] polyA additional signal, [4] glucocorticoid sensitive factor (GRE). Since the genes in the HBV genome are transcribed on the three HBV mRNA transcripts, respectively, there should be at least three RNA polymerase II promoters at the proximal 5′ end of each transcript in the viral genome, although these promoters The gene sequences are not known, but these promoters are apparently present within the encoded protein sequence. The enhancer (ENH) is located in the polymerase gene; the polyA additional signal is located in the CORF; and the GRE is located in the SORF and polymerase genes. GRE is a DNA fragment of a hormone receptor structure that, when combined, increases the level of transcription of a known gene.

GRE has many enhancer features: [1] is a factor that acts as a cis, [2] acts in both directions of transcription, [3] can function at different distances from the genes it regulates.

It can be seen from the above that the HBV genome is structurally strict and tissue efficient, and is rare in known viruses. HBVDNA not only has its unique structure, but its DNA replication process is also very special. When HBV DNA enters the host cell, it first becomes a complete closed-loop double-stranded DNA, and the negative strand is used as a template to synthesize a full-length “+” strand RNA (called pre-genomic RNA). The “+” strand RNA is packaged in immature core-like particles, and a DNA polymerase and a protein are also packaged in the particles. In the granule, the “+” strand RNA is used as a template to catalyze the synthesis of “-” strand DNA by reverse transcriptase. The specific mechanism is unclear, and may be similar to the replication of adenoviral DNA, because at the 5′ end of the “-” strand DNA. There are also proteins that are covalently bound. The synthesis of “+” strand DNA is polymerized and extended with the negative strand DNA as a template and a piece of RNA as a primer, and the core-like virus particles also become mature virus particles in the process. At this time, the positive strand DNA is still not synthesized, resulting in different lengths of the two DNA strands of the viral genome.

About us

Through nearly ten years’ hard working and depend on our professional work team, we are proud of satisfying the needs of our clients both at home and abroad, which across more than 50 countries and districts. We always devote ourselves to providing you with the best and professional service. Our products including: Total RNA Sequencingpacific biosciencessingle cell transcriptomicssingle cell genomics,etc.

Oligosaccharide fructose is a sugar cane triose

Oligosaccharide fructose, also known as cane oligosaccharide, is a sugar cane triose, cane sugar and sugar cane fruit produced by combining 1 to 3 fructosyl groups through a β(2-1) glycosidic bond with fructose groups in sucrose. a mixture of sugars and the like. There are about 60-70 grams of inulin in 100 grams of dry weight Jerusalem artichoke. Inulin is a fructan linked by a linear β-2,1-glycosidic chain with a sucrose group at its end. Therefore, the inulinase hydrolysis is used as the raw material of the Jerusalem artichoke powder, and the oligofructose syrup is finally obtained after purification.

Ingredient

International labeling

The oligofructose refers to a carbohydrate formed by the linkage of 2 to 5 fructosyl groups as a chain link, a terminal group having a glucosyl group as a chain, and a fructose-based→fructose linkage as a main skeleton. That is to say, 1 to 4 fructose groups are formed by β-2,1 linkage to the D-fructose group of sucrose, which can form GF2, GF3, and GF4. And a mixture of cane fruit hexose (GF5). Commercial oligofructose generally also contains a small amount of sucrose, fructose, and glucose. (Refer to the definition in the national standard currently under development)

Active substance

Oligofructose is a natural active substance. The sweetness is 0.3-0.6 times that of sucrose. It not only maintains the pure sweet taste of sucrose, but also has a sweeter taste than sucrose. It is a new type of sweetener with regulating the intestinal flora, proliferating bifidobacteria, promoting calcium absorption, regulating blood lipids, immune regulation, anti-caries and other health functions. It is known as the most potential new generation additive after the antibiotic era. – Biomass-promoting; known as Protoxin (PPE) in France, it has been used in a variety of foods such as dairy products, lactic acid bacteria beverages, solid beverages, candy, biscuits, bread, jelly, and cold drinks.

 

Benefits
Health effect
In addition to the physicochemical properties of oligofructose, the most striking physiological characteristic is that it can significantly improve the proportion of microbial population in the intestine. It is activating proliferative factor of Bifidobacterium in the intestine, which can reduce It inhibits the production of spoilage substances in the intestines, inhibits the growth of harmful bacteria, regulates the balance in the intestines, promotes the absorption and utilization of trace elements iron and calcium, prevents osteoporosis, reduces liver toxins, and produces anticancer in the intestines. The organic acid has a remarkable anti-cancer function; and the taste is pure and sweet, with a fat-like aroma and a refreshing smoothness. In recent years, the products of oligofructose have been popular in the health care products market such as Japan, Europe and America.

The syrup is a colorless or light yellow, transparent viscous liquid with a oligofructose fragrance, soft and refreshing sweetness, no odor, no foreign impurities.
The powdered sugar is white or yellowish amorphous powder (the particles are white or yellowish amorphous particles), the sweetness is soft and refreshing, the oligofructose is fragrant, no odor, no foreign impurities.

Six functions

  1. Low calorific value, because oligofructose can not be directly digested and absorbed by the human body, it can only be absorbed and utilized by intestinal bacteria, so its caloric value is low, it will not lead to obesity, and indirectly has weight loss. It is also a good sweetener for people with diabetes.
  2. Because it cannot be used by oral bacteria (referred to as Smutans), it has anti-caries effect.
  3. Proliferation of the beneficial bacteria in the intestine. Oligofructose selectively proliferates beneficial bacteria such as Bifidobacterium and Lactobacillus in the intestine, which makes the beneficial bacteria occupy an advantage in the intestine, inhibits the growth of harmful bacteria, and reduces toxic substances (such as endotoxin, ammonia, etc.) The formation of the intestinal mucosal cells and liver protection, thereby preventing the occurrence of diseased intestinal cancer, enhance the body’s immunity.
    4. Reduce serum cholesterol and triglyceride levels.
  4. Promote the absorption of nutrients, especially calcium. Ingestion of oligofructose can increase the absorption of calcium ions by organisms. This phenomenon has received more and more attention, and some human clinical experiments have been carried out. For adolescents, inulin rich in oligofructose has been shown to have a positive effect on bone health by enhancing calcium absorption and increasing bone density. For women after menopause, oligofructose-rich inulin has been shown to improve mineral absorption and improve bone health. In addition, clinical studies have indicated that supplementation of inulin rich in oligofructose can help improve mineral absorption and affect bone turnover markers in menopausal women.
  5. Prevention of diarrhea and constipation.

 

Mechanism of action
Oligofructose is a water-soluble dietary fiber that can reduce serum cholesterol and improve lipid metabolism after long-term use, as confirmed by animal and human experiments. Oligofructose has the following physiological functions: it is utilized by beneficial bacteria such as bifidobacteria, that is, it only proliferates 10 to 100 times, and bifidobacteria (pathogenic bacteria) have the effect of two-way regulation. After the human ingestion of oligofructose, the number of beneficial bacteria in the body can inhibit the growth and reproduction of exogenous pathogenic bacteria and intestinal decaying bacteria such as Salmonella, reduce the growth and accumulation of intestinal decay substances, and promote intestinal peristalsis. Prevent constipation and diarrhea. Oligofructose is an excellent water-soluble dietary fiber, which can effectively reduce the amount of serum cholesterol, triglyceride and free fatty acids. It is good for a series of cardiovascular diseases such as hypertension and arteriosclerosis caused by high blood lipids. Improve the role. Oligosaccharide fructose is fermented by bacteria in the large intestine to produce L-lactic acid, which can dissolve minerals such as calcium, magnesium and iron, and promote the absorption of minerals by the human body.
Experiments have confirmed that oligofructose promotes calcium absorption rate of 70.8%. Therefore, oligofructose can promote growth and prevent osteoporosis. At the same time, it can promote the natural formation of vitamins B1, B2, B3, B6, B12 and folic acid, thereby improving the body’s metabolism level, improving immunity and disease resistance. Prevent and improve skin diseases caused by poisoning in the body, prevent facial sores, dark spots, freckles, acne, and age spots, so that the skin is bright and the aging is slowed down. After absorption of oligofructose, bifidobacteria rapidly proliferate, inhibiting the action of spoilage bacteria such as Escherichia coli, Salmonella and Clostridium, reducing the formation of toxic metabolites (such as guanidine and nitrosamine), and rapidly toxic metabolism. Excretion of the body, reduce the burden on the liver, protect the liver, prevent various chronic diseases, cancer and other effects, oligofructose is rarely decomposed by the stomach acid and enzymes in the digestive tract, extremely difficult to be absorbed by the body. It has been determined that the caloric value of oligofructose is less than 1.5 Kcal/g, and the caloric value of sucrose is 4.6 Kcal/g. Therefore, it is ideal for low-calorie functional sweetness after ingesting oligofructose. Oligof fructose can not be used by Streptococcus mutans to form insoluble glucan to provide oral microbial deposition \ acid and corrosion sites (tartar), thus preventing dental caries.

Ingredient study
The proliferation of nine strains of Lactobacillus was studied by in vitro experiments with three different degrees of polymerization of garlic fructan Garlic Fructans (GF1, GF2, GF3). The in vitro fermentation experiment of Lactobacillus was carried out with garlic fructan as the sole carbon source, and compared with two commercial prebiotics-Fructo-oligos aecharides (FOS) and inulin (Inulin), the pH of the fermentation broth was 48h. And the Prebiotic Index (PI) value of the probiotic index was used as an evaluation index to evaluate the prebiotic effect of garlic fructan. The results showed that the PI value of GF1 to nine strains of Lactobacillus reached 0.6 or more, and the pH of the fermentation broth decreased significantly, which was better than FOS and Inulin, while the PI value and pH of GF2 and GF3 fermentation broth decreased with FOS and Inulin. Basically equivalent, indicating that all three kinds of garlic fructan have a certain prebiotic effect, and the lower the degree of polymerization, the stronger the prebiotic function. The results also showed that the P-type of Lactobacillus paracasei for the five fructans (GF, FOS, Inulin) reached 0.6 or more, and the same type of fermentation produced a large amount of lactic acid, indicating that its utilization of fructan proliferation and acid production is outstanding.

About us

We provide a wide range of high quality normal human and animal cells, cell culture medium and reagents, FISH probes, tissue arrays, microorganisms and equipments. In addition, we also offer series of related services including cell services, biosample services and histology services for the researcher to make their project better and faster. Here are some products:FSTL5FTCDFTLFTO,etc.

Peptide Synthesis Is a Solid Phase Synthesis Sequence

 

Peptide synthesis is a solid phase synthesis sequence generally synthesized from the N-terminus (amino terminus) to the C-terminus (carboxy terminus). Past peptide synthesis is carried out in solution called liquid phase synthesis. Since Merrifield developed the solid phase peptide synthesis method in 1963, it has been continuously improved and perfected. Today, the solid phase method has become a common technique in the synthesis of peptides and proteins, showing the advantages unmatched by classical liquid phase synthesis, thus greatly reducing the difficulty of purification of each step of the product. Peptide synthesis is generally divided into two types: solid phase synthesis and liquid phase peptide synthesis.

Definition of polypeptide

A polypeptide is a biologically active substance related to various cellular functions in an organism. Its molecular structure is between amino acids and proteins, and is a compound obtained by combining a plurality of amino acids in a certain order by peptide bonds. Polypeptides are a general term for biologically active substances involved in various cellular functions in living organisms, and are often used in functional analysis, antibody research, and especially in drug development.

In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS), which was the first choice for peptide synthesis due to its convenient and rapid synthesis, and brought about a revolution in the organic synthesis of peptides and became an independent discipline. – Solid phase organic synthesis, the invention of solid phase synthesis also promotes the automation of peptide synthesis. The world’s first true peptide synthesizer appeared in the early 1980s.

Peptide synthesis technology

In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS), which was the first choice for peptide synthesis due to its convenient and rapid synthesis, and brought about a revolution in the organic synthesis of peptides and became an independent discipline. – Solid phase organic synthesis, the invention of solid phase synthesis also promotes the automation of peptide synthesis. The world’s first true peptide synthesizer appeared in the early 1980s.

Liquid phase synthesis

The synthesis is carried out step by step based on the repeated addition of a single N-alpha protected amino acid to the growing amino component, usually starting from the C-terminal amino acid of the synthetic chain, followed by the attachment of a single amino acid by DCC, mixing with charcoal anhydride, or N- The carboxy anhydride method is implemented. The Carbodiimide method involves the use of DCC as a linker to link N- and C-protected amino acids. Importantly, this ligation reagent facilitates the shrinkage of the N-protected amino acid self-carbon group and the C-protected amino acid free amino group to form a peptide chain, while producing N, N?/FONT>-dyaylcohercylurea by-product. However, this method is affected by the side reactions which cause racemization or the formation of 5(4H)-oxaylones and N-acylurea in the presence of a strong base. Fortunately, these side reactions can be minimized, but they cannot be completely eliminated. The method is to add a linking catalyst such as HoSu or HoBT. In addition, this method can also be used to synthesize an active ester derivative of an N-protected amino acid. The resulting active ester will spontaneously react with any other C-protected amino acid or peptide to form a new peptide.

Type of peptide synthesizer

Peptide synthesizers are instruments used to synthesize peptides. Because the steps of peptide synthesis are cumbersome and time consuming, many companies have developed automated peptide synthesizers. Generally divided into two categories, one is for a small number of production applications with a large amount of synthesis, the typical representative is the ABI336 type peptide synthesis instrument of American ABI Company, the reactor rotation mode is different from the previous two generations of peptide synthesis instruments. That is, the reactor is relatively fixed above, and the lower side is rotated 360 degrees rapidly, which drives the solid-liquid two phases in the reactor to spiral upward from the bottom to reach the top of the reactor; another type of peptide synthesizer is aimed at drug development. Scientific research applications such as genetic research, such as ResPep SL and MultiPep RS series from Intavis AG of Germany, can provide both solid phase synthesis and membrane synthesis applications, and can automatically assist customers in mapping and high-throughput peptides. Synthesis work.

Classification of peptide synthesizers

The advent of peptide synthesizers has greatly contributed to the development of peptide science. Conversely, with the development of peptide science, scientists have put forward higher requirements for synthesizers, which has led to the development of synthesizers. At present, there are many varieties of peptide synthesizers, which can be divided into micrograms, milligrams, grams and kilograms from the synthesis amount; from the functional points, they can be divided into research type, small test type, medium Trial type, general production type and GMP production type; from the degree of automation, can be divided into fully automatic, semi-automatic and manual; from the channel, can be divided into single channel and multi-channel; From the technical point of view, it can be divided into the first generation, the second generation, and the third generation; and so on.

Operating principle

The peptide synthesizer uses solid phase synthesis as the reaction principle, and the amino acid is continuously added, reacted, synthesized and operated in a known order (sequence, generally from C-carboxyl end to N-terminal-amino end) in a closed explosion-proof glass reactor. The polypeptide carrier is finally obtained. The solid phase synthesis method greatly reduces the difficulty of purification of each step of the product. In order to prevent the occurrence of side reactions, the side chains of the amino acids participating in the reaction are protected. The carboxy terminus is free and must be activated prior to the reaction. There are two solid phase synthesis methods, namely Fmoc and tBoc. Because Fmoc has many advantages over tBoc, it is mostly synthesized by Fmoc method. However, for some short peptides, tBoc is still used by many companies because of its high yield.

The specific synthesis consists of the following cycles:
1) Deprotection: Fmoc protected columns and monomers must be treated with a basic ion (piperidine) to remove the protecting group of the amino group.
2) Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to form a peptide bond. In this step, a large amount of super-concentration reagent is used to drive the reaction to completion. Cycle: The two steps of the reaction are repeated until the synthesis is complete.
3) Elution and deprotection: The polypeptide is eluted from the column and its protecting group is eluted and deprotected by a deprotecting agent (TFA).

About us

Creative Peptides is specialized in the process development and the manufacturing of bioactive peptides. We are dedicated to offering custom peptide synthesis, process development, GMP manufacturing as well as catalog products for customers in industry and research area.here are some our products like: Peptide Modification, De novo Peptide Design, Long Peptides Synthesis, Peptide Drug Discovery, etc.

Magnetic Particle: Familiar or Strange?

When it comes to magnets, we are no strangers to everyone. In everyone’s mind, magnetic particle is the magnetic particle that falls off the magnet, but this is only part of the real face of the magnetic particle. The world of magnetic particle can be much more than just as simple as you think. Let me reveal the magical side of themagnetic particle you are familiar with!

What is the standard definition of magnetic particle?

Magnetic particle is a hard magnetic single domain particle. It is made into a magnetic paste with a binder, a solvent, etc., and coated on the surface of a plastic or metal substrate to form a magnetic recording material such as a magnetic tape, a magnetic disk, or a magnetic card. Commonly used magnetic particles are two major types of metal magnetic particles: oxide magnetic particle and metal magnetic particle.

Various uses of magnetic particle

Excellent performance in inspection work

Magnetic particle testing is a magnetic flaw detection method based on the principle of magnetic flux leakage. After the ferromagnetic material and the workpiece are magnetized, due to the discontinuity, the magnetic field lines on the surface of the workpiece and the near surface are locally distorted to generate a leakage magnetic field, the magnetic particle applied on the surface of the workpiece is adsorbed to form a magnetic field visible under suitable illumination, and traces, showing the location, shape and size of the discontinuities.

Nano Fe3O4 is a common material for magnetic flaw detection and belongs to nanoparticles. There are eight main advantages:

v Characteristics of high magnetic permeability, low coercivity, and low remanence;

v Good dispersibility, not easy to agglomerate, strong magnetic;

v Good magnetic permeability and good mobility;

v Mixing of nanomaterials with different dimensions can improve the performance of magnetic particle testing process;

v It can be combined with organic materials to modify nano magnetic materials;

v It can achieve consistent particle size and meet the needs of special testing;

v The detection rate of particle crack is high, and it has good performance for small crack detection;

v Low manufacturing costs.

n Advantages and limitations of magnetic particle testing

Advantages of magnetic particle detection method:

Ø The shape, position and size of the defect can be visually displayed, and the properties can be roughly determined;

Ø This method can detect cracks with a minimum length of 0.1 mm width and micron order, which is very High sensitivity;

Ø It is not restricted by the size and shape of the test piece;

Ø The method is simple to use, less polluted, and low in cost, and the detection speed is fast; 5 can be repeatedly tested.

Limitations of the magnetic particle detection method:

Ø It can only be used for ferromagnetic materials;

Ø it is only for the surface and near surface defects of the material;

Ø it is subject to the magnetization direction, and the defect direction is approximately parallel to the magnetization direction or the defect and the surface of the workpiece When the angle is less than 200, it is difficult to find defects.

Ø It will be restricted by the geometry, and it is easy to produce non-correlated display; 5 If the surface of the workpiece has a cover layer, it may affect the detection result of the magnetic particle.

Electric vehicle magnetic particle brake control technology

The magnetic particle brake is a kind of magnetic particle filled in the working gap between the stator and the rotor, the magnetic particle is combined by the electromagnetic attraction force and the friction between the magnetic particle and the working surface transmits power and movement, and can control the torque adjustment system. The physical object of the magnetic particle brake is shown on the right.

Magnetic particle brake has the advantages of fast reaction speed, no pollution, no noise, energy saving, etc. It is a multi-purpose, superior performance automatic control component. Under the premise of ensuring safety, the advantage of electric vehicle magnetic particle brake is to improve energy utilization, as well as reduce mechanical wear of mechanical and hydraulic braking modes, achieve more precise braking control, and reduce traditional vehicles. Braking thermal decay caused by temperature rise during braking.

A good helper to optimize water quality

With the acceleration of industrialization and urbanization in China, a large amount of untreated industrial sewage and domestic sewage are directly discharged into the river, the eutrophication of water bodies is becoming more and more serious, anaerobic bacteria are multiplied, organic matter is spoiled, decomposed, fermented, and finally black.

The test shows that it is feasible to purify the black odor river water by adding magnetic particle. Compared with other flocculants, it has the characteristics of short sedimentation time, stable treatment effect, green environmental protection, no secondary pollution, etc. Controlling eutrophication of water bodies is a simple and efficient technique for polluting river water.

Ø The combination method of laying the magnet at the bottom further shortens the processing time, improves the processing efficiency, increases the treatment effect, can effectively reduce the volume of the treatment structure, and greatly reduces the investment cost.

Ø The magnetic particle can be mostly recovered, and the magnetic particle is reused, the dosage of the medicine is reduced, and the running cost is reduced.

Ø The technology can be applied to the treatment of high suspended solids production wastewater such as paper making, fiberboard processing, coal washing, desulfurization wastewater, graphite, marble processing.

Ø The disadvantage of this method is that it has a removal effect on dissolved pollutants, and can be combined with bio-ecological technology to further improve the treatment efficiency of black odorous water.

The future of magnetic particles

Now, do you have a new understanding of magnetic particle? It must be said that in addition to magnetic particle, magnetic beads and magnetic microspheres are also part of this family; they are showing their unique charm for human biology and medicine. At this stage, the basic theoretical research of magnetic particle testing has been relatively mature, and we believe that the future magnetic particle will have a broader application prospect!

References

[1] Experimental Study on Removal of Pollutants with Magnetic Powder from Black – and – Malod orous Water Body. Jouranal of Taishan University. Vol.39 NO.6 Nov. 2017.

[2] On Application of Magnetic Powder Detection in the Pressure Vessel Inspection. 1006-4311(2014)09-0019-02.

The Unique Charm of Magnetic Beads

What is a magnetic bead?

The magnetic bead is called a ferrite bead filter and is an ultra-small magnetic component that has been introduced in recent years. It is made by sintering a ferrite material (or amorphous alloy) with a wire at a high temperature. Magnetic particle is a much smaller magnetic material than magnetic beads, but its properties are quite different from magnetic beads. Below I will take you to appreciate the charm of magnetic beads!

The main features of the magnetic beads:

  • Magnetic beads have the characteristics of high frequency loss, high resistivity and high magnetic permeability, and can suppress noise interference in an extremely wide frequency band. By connecting the magnetic beadsin series in the path of the signal or power supply, the series mode noise interference can be effectively suppressed.
  • The magnetic beads can be equivalent to a series circuit of inductance and resistance, and the equivalent inductance and equivalent resistance are proportional to the length of the magnetic beads. In the DC or low frequency band, the magnetic beads exhibit a very low inductive reactance and do not affect the signal transmission on the data line or signal line.
  • A magnetic bead is measured by the impedance it produces at a certain frequency, and its unit is Q, not H.
  • Magnetic beads have an absorption effect on high-frequency components, and are therefore also referred to as absorption filters. In contrast, the role of the common mode inductor in the EMI filter is to reflect electromagnetic interference back to the signal source, which is a reflective filter.
  • Magnetic beads are devices that convert high-frequency energy into eddy currents and dissipate them in the form of heat. For short, energy-consuming devices, the eddy current loss is proportional to the square of the noise frequency. The common inductor is an energy storage component, referred to as an energy storage component. The magnetic bead has a maximum operating frequency of 1 GHz, and the operating frequency of the inductor generally does not exceed 50 MHz.
  • Magnetic beads can suppress the generation of switching noise. It is an active suppression of high frequency interference. This is because the magnetic beads are connected to the main circuit (the output circuit) that generates the spikes, and the inductance is used to reduce the rate of rise of the spike current, so it is called “active suppression type”. The electromagnetic interference filter can only passively suppress interference, so it is called “passive suppression type”, which is the fundamental difference between the two.
  • It is allowed to use a plurality of magnetic beads in series or in parallel. Usually, the beads should be installed close to the source of the interference.
  • Magnetic beads can be used not only in high-frequency switching power supplies, electronic measuring instruments, and various circuits with strict requirements on noise, but also in digital home appliances such as mobile phones, DVDs, and digital video cameras. Chip beads eliminate RF interference in the transmission line. In electromagnetic compatibility (EMC) design, magnetic beads are a kind of magnetic component commonly used to suppress high frequency electromagnetic interference.

Several important applications of magnetic beads

  • “Universal” immunomagnetic beads

Immunomagnetic beads are a new immunological technique developed in recent years. It combines the unique advantages of curing reagents with the high specificity of immunological reactions. It is based on immunology and penetrates into various fields such as pathology, physiology, pharmacology, microbiology, biochemistry and molecular genetics. It is in immunoassay and cell separation. Biomacromolecule purification and molecular biology have been widely used. Immunomagnetic beads can be mainly used in the following aspects:

  • Cell separation.
  • Purification of biological macromolecules.
  • The application of molecular biology.

  • Application in nanopore DNA sequencing

Since 1996, the use of nanopores as sensors to analyze DNA sequences has become a hot research area. The principle of the method is that when the DNA molecule passes through the nanopore, local clogging of the nanopore is caused, thereby realizing the change of the ionic current of the vias, and the information of the transwell DNA sequence is obtained by the change of the ionic current. However, in order to overcome the entropy resistance of DNA, in order to drive DNA, it is necessary to apply a large current, which will cause the DNA to pass through too fast, which will reduce the temporal and spatial resolution of DNA vias and increase the noise. Therefore, reducing DNA via velocity is a key technology for achieving nanopore determination of DNA sequences. Tests have shown that DNA can be slowed down by attaching DNA to well-beaded beads or magnetic beads and controlling the beads by light or magnetic enthalpy.

  • Refuge for insomnia patients

You certainly can’t think of a small magnetic bead that will bring good news to patients with insomnia. The magnetic bead acupoint method is simple to operate, stimulates by ear acupuncture, regulates the corresponding organ function, and plays a role of conditioning and health care. It is a non-invasive, non-adverse-response, safe, non-medical and traditional Chinese medicine health care measure. The majority of patients hospitalized in the chest are elderly. Compared with the application of sedative and hypnotic drugs, the magnetic bead acupoint method is safer and has no side effects. It is an effective drug replacement therapy, which promotes sleep and rehabilitation of patients and improves patients. The quality of life has given full play to the advantages of traditional Chinese medicine nursing technology.

Small Quantity Plastic Containers – Buying the Right Amount of Containers for Your Needs

If you’re a person who is in need of clear plastic containers but you’re overwhelmed with the choices facing you, here are a few ideas to help. First off, the reason you need these containers will determine which type of container you should end up with.

You may think that if you’re looking to purchase from a plastic manufacturing company that you’ll have to order a huge amount of containers. The thought of that may be preventing you from ordering what you need. If you don’t need a large quantity of containers, your best bet would be to find a plastics manufacturer who specializes in small quantity orders.

You don’t want to use your company’s budget on unnecessary items. So finding just the right amount of containers would be a great benefit. The advantage of ordering in small quantities is, you’ll get to decide what will be the right amount of plastic containers for your needs.

Find a company that offers high clarity, high impact clear PVC plastic. PVC is a great general purpose plastic. Make sure the plastic containers you’re purchasing are FDA approved, food grade plastic. With this you can be assured food will be safe if it comes in contact with the plastic. There won’t be any harmful chemicals. With this in mind you’ll be able to display candies, nuts, and snack items, such as pretzels or beef jerky.

Also look for containers that have wide mouth openings. Then if you’re displaying merchandise it will be easy for potential customers to handle the items. If a customer holds something in their hands, they’re more likely to buy it. So find a container that makes this step less cumbersome for your customers. It’s best to find a company that offers lids as well because transporting your merchandise should be worry free.

Let’s say you’re a business owner looking to set up displays of your merchandise at trade shows or your shop. Clear plastic containers would be perfect for displaying a variety of items specific to your business. For instance if you’re in the hardware business you could take advantage of containers that highlight nuts, bolts, screws, nails, small flashlights or tape measures.

If you’re in the beauty business, plastic containers are perfect for displaying nail polish, lipstick, fingernail files, lotions, and small hair care products such as brushes or combs.

If you’re looking to sell products at a craft fair or school carnival, plastic containers would do the job. They could hold whistles, stickers, costume jewelry, pencils, erasers, key chains or small toys.

Some times in business, deadlines sneak up on us and we panic. To help alleviate this panic it’s best to find a plastic manufacturer who can ship most orders in 24 hours.
Also look for a company that has drop ship capabilities.

If you’re running your own business you know that your business has exclusive needs. Your merchandise is your business, how you display your merchandise is part of your image. Find a clear plastic manufacturer who offers different sizes and shapes of containers.

Running your business can be made easier with the right people in your corner. Finding a reliable, experienced plastic manufacturer can help with one of the biggest components of your business, displaying your merchandise. With the right display, overall sales could improve. The right clear plastic container, and the right amount of clear plastic containers are important to your business.

Article Source: https://www.china-plasticmolding.com/

The Methods of Whole Genome Sequencing

Overview of Whole Genome Sequencing

The genome of each individual organism contains its entire genetic information. Whole genome sequencing technology can comprehensively and accurately analyze entire genomes, thereby breaking the information contained in it and revealing the complexity and diversity of the genome. The emergence of whole genome sequencing technology is a revolutionary advancement in all areas of life sciences. Whole genome sequencing can detect variants, including single-nucleotide variants, insertions/deletions, copy number changes, and large scale structural variants. Whole genome sequencing can be classified into de novo and resequencing depending on whether there is a reference genome. If there is a reference genome, genome assembly will become more easy and rapid.

  1. Two Classic Approaches for Sequencing Large Genomes

In the early 80s, Sanger successfully completed a whole genome sequencing of the lambda phage by using the shotgun method, and the method was successfully applied to the larger virus DNA, the organelle DNA, and the sequencing of the bacterial genome DNA. Shotgun sequencing is the classic strategy for whole genome sequencing. The shotgun sequencing strategy provides a technical guarantee for large-scale sequencing. The technology first randomly interrupts a complete target sequence into small fragments, sequenced separately, and then splicing them into a consistent sequence by using the overlapping relationships of these small fragments. It mainly includes two methods: one is hierarchical shotgun sequencing (clone-by-clone method) and the other is whole genome shotgun sequencing.

  • Clone-by-clone sequencing

This method was once adopted by the HGP consortium. This method can generate high density maps, making the genome assembly easier. It generally includes four steps, preparation of BAC clone library, preparation of clone fingerprint, BAC clone sequencing, and sequence assembly. However, this method is time-consuming and costly, so it is seldom used at present.


Figure 1. Steps involved in the clone-by-clone sequencing.

WGS generally involves six steps, isolation of genomic DNA, random fragmentation of genomic DNA, size selection using electrophoresis, library construction, paired-end sequencing (PE sequencing), and genome assembly. Two different sizes of DNA fragments including longer insert (2-2.5 kb) and short insert (0.5-1.2 kb) are selected from the agarose gel. While the long inserts are cloned in phage or socmid vectors, the short inserts are cloned in plasmid vectors. The short insert clone library is used for sequencing from both the ends. Since large numbers of clones are sequenced, each of the genomes will be covered more than 10 times. Long insert clones can be used to increase the efficiency of genome assembly.

Advantages:

  • Does not require genome maps.
  • Less time consuming
  • Money-saved

Disadvantages:

  • Genome assembly for eukaryotic genomes is difficult due to abundant repetitive sequences
  • Genome sequencing using this method is not accurate.
  1. NGS Accelerates WGS

Unlike clone-based library approaches, next-generation sequencing platforms utilize a dramatically simplified method of library construction, which has simplified and accelerated the whole genome shotgun sequencing. In generally, genomic DNA is first randomly fragmented using sonication or nebulization, and then are ligated to a platform-specific set of double-stranded adapters to generate a shotgun library. Subsequently, these library fragments can be amplified in situ by hybridization and extension from complementary adapters which are covalently attached to the surface of a glass microfluidic cell or a small bead (depending on the sequencing platform). All NGS instruments utilize a microfluidic device to contain the amplified fragments of the shotgun library, followed by an imaging step that collects data from fragments being actively sequenced.

We will take the Illumina sequencer as an example to illustrate the workflow of WGS based on high-throughput sequencing.

  • Construction of Sequencing Library

The genome is first prepared, and then the DNA is randomly fragmented into hundreds of bases or shorter fragments with specific adapters at both ends. If the transcriptional group is sequenced, the library construction is a bit more troublesome. After the RNA fragmentation, it needs to reverse to cDNA, then add the connector, or reverse the RNA to the cDNA first, then fragment and add the joint. The size of the fragment (insert size) has an impact on the subsequent data analysis and can be selected according to needs. For genome sequencing, several different insert sizes are usually chosen to get more information when assembling.

  • Surface Attachment and Bridge Amplification

The reaction of Solexa sequencing is carried out in a glass tube called flow cell, and flow cell is subdivided into 8 Lanes, each of which has a number of fixed single strand joints on the inner surface of each Lane. The DNA fragment of the joint was transformed into a single strand and combined with the primers on the sequencing channel to form a bridge like structure for subsequent preamplification.

  • Denaturation and Complete Amplification

The unlabeled dNTP and the common Taq enzyme were added for solid phase bridge PCR amplification, and the single-stranded bridge sample was amplified into a double-stranded bridge fragment. By denaturation, a complementary single strand is released and anchored to the nearby solid surface. By continuously cycling, millions of clusters of double-stranded analytes will be obtained on the solid surface of the Flow cell.

  • Single Base Extension and Sequencing

Four fluorescently labeled dNTPs, DNA polymerases, and linker primers were added to the sequenced flow cells for amplification. When each sequencing cluster extends the complementary strand, each fluorescent labelled dNTP is added to release the corresponding fluorescence. The sequencer obtains sequence information of the fragment to be tested by capturing a fluorescent signal and converting the optical signal into a sequencing peak by computer software. The read length is affected by a number of factors that cause signal attenuation, such as incomplete cutting of fluorescent markers. As the length of the reading increases, the error rate will also increase.

  • Data Analysis

This step is not strictly a part of the sequencing process, but it only makes sense through the work in front of this step. The raw data obtained by sequencing is a sequence of only a few tens of bases in length, and the contigs that assemble these short sequences through bioinformatics tools are even the framework of the entire genome. Alternatively, these sequences are aligned to an existing genome or a similar species genome sequence, and further analyzed to obtain biologically meaningful results.

  1. Application of Third-generation Sequencing Sequencing in Whole Genome Sequencing

Although next-generation sequencing has enabled population-scale analyses of small variants, it’s difficult to identify larger structural variations. Further, de novo assembly using next-generation sequencing are often of lower quality compared with those using older and more expensive methods. The single-molecule sequencing technologies can get over these difficulties, which can span nearly entire chromosome arms and are not sensitive to GC content. Third-generation sequencing technologies have been used to produce highly accurate de novo and reference assemblies for microorganisms, plants, animals, and humans, enabling new insights into evolution and sequence diversity.

If you are interested in our genomics services, please feel free to contact our scientists.

References:

  1. Bentley D R. Whole-genome re-sequencing. Current Opinion in Genetics & Development, 2006, 16(6):545-552.
  2. Fuentespardo A P, Ruzzante D E. Whole-genome sequencing approaches for conservation biology: advantages, limitations, and practical recommendations. Molecular Ecology, 2017, 26(20):5369.
  3. Batzoglou S, Berger B, Mesirov J, et al. Sequencing a genome by walking with clone-end sequences (abstract):a mathematical analysis// International Conference on Computational Molecular Biology. DBLP, 2000:45.
  4. Sanger F ,, Coulson A R, Hong G F, et al. Nucleotide sequence of bacteriophage lambda DNA. Journal of Molecular Biology, 1982, 162(4):729-73.
  5. Kawarabayasi Y, Sawada M, Horikawa H, et al. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. Dna Research, 1998, 5(2):55.
  6. Kaneko T, Sato S, Kotani H, et al. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. Dna Research, 1996, 3(3):185-209.
  7. Myers E W, Sutton G G, Delcher A L, et al. A Whole-Genome Assembly of. Science, 2014.
  8. Siegel A F, Engh G V D, Hood L, et al. Modeling the Feasibility of Whole Genome Shotgun Sequencing Using a Pairwise End Strategy. Genomics, 2000, 68(3):237.
  9. White O, Fraser C M. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science, 1999, 286(5444):1571-1577.
  10. May B J, Zhang Q, Li L L, et al. Complete genomic sequence of Pasteurella multocida, Pm70. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(6):3460-3465.
  11. Ginsburg G S, Willard H F. Genomic and personalized medicine. Academic Press, 2008.

Source: https://www.cd-genomics.com/resourse-The-Methods-of-Whole-Genome-Sequencing.html