What are the nucleic acid extraction methods?

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The principle of nucleic acids extraction firstly guarantees the integrity of the primary structure of nucleic acid, and secondly eliminates the contamination of other molecules. When extracting, attention should be paid to shorten the extraction time, reduce the degradation of nucleic acid by chemical and physical factors, mechanical shearing force and high temperature, and prevent the biological degradation of nucleic acid. What are the methods of nucleic acid extraction?

 

1.Concentrated salt method

(1). Using the different solubility of RNP and DNP in the electrolytic solution to separate the two, the common method is to extract the DNP mucus obtained by extracting sodium chloride with 1M sodium chloride and shaking together with chloroform containing a small amount of octanol to make the emulsification and centrifugation to remove Protein. At this time, the protein gel stays between the water phase and the chloroform phase, while the DNA is in the upper water phase. The sodium salt of DNA can be precipitated with 2 times the volume of 95% ethanol.

(2). You can also use 0.15 M NaCL to repeatedly wash the cell crushing solution to remove RNP, then extract deoxyribonuclein with 1 M NaCL, and then remove the protein by chloroform—isoalcohol method. Comparing these two methods, the second method may reduce nucleic acid degradation.

 

(3). When extracting DNA with dilute hydrochloric acid solution, adding an appropriate amount of detergent, such as SDS, can help separate protein and DNA. In the extraction process, in order to inhibit the degradation of DNA by DNase in the tissue, sodium citrate was added to the sodium chloride solution as a melting agent for metal ions, usually 15M NaCL, 0.015M sodium citrate, and called SSC solution, to extract DNA.

 

2.Anionic detergent method

Detergents such as SDS or sodium xylate are used to denature proteins, and DNA can be extracted directly from biological materials. Because DNA and protein in cells are often combined by electrostatic attraction or coordination bonds, anionic detergents can destroy this. A kind of valence bond, so anionic detergents are often used to extract DNA.

 

3.Phenol extraction method

Phenol acts as a protein denaturant, and at the same time inhibits the degradation of DNase. When the homogenate is treated with phenol, the bond between the protein and DNA is broken, and the surface of the protein molecule contains many polar groups that are compatible with phenol, and the protein molecule is soluble in phenol. Phase, while DNA is soluble in the water phase. After centrifugation and layering, take out the water layer, repeat the operation several times, and then combine the DNA-containing water phase, and use the alcohol-insoluble property of nucleic acid to precipitate the DNA with ethanol. At this time, DNA is a very viscous substance, and it can be taken out by wrapping it around in a ball with glass. The characteristic of this method is to keep the extracted DNA in its natural state.

 

4. water extraction method

Utilizing the nature of nucleic acid to dissolve in water, after breaking tissue cells, remove RNA with low-salt solution and dissolve the precipitate in water to fully dissolve DNA in water. After centrifugation, collect the supernatant and add solid sodium chloride to the supernatant. To 2.6M, add 2 times the volume of 95% ethanol, stir out immediately, and then wash with 66%, 80%, and 95% ethanol and acetone respectively.

The above gives a brief introduction to what are the methods of nucleic acid extraction. Ascend provides professional and complete casting services for nucleic acid extraction products.

isolation and purification of nucleic acids method

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Nucleic acid is an important carrier of genetic information and an important biological information molecule. It plays a key role in the process of people’s biological research. At present, there are many methods for isolation and purification of nucleic acids . The most commonly used is (iso)guanidine thiocyanate What is the one-step method of phenol-chloroform, this method is briefly introduced below.

Due to the influence of RNase, in order to obtain a complete RNA molecule, it is necessary to inactivate the activity of intracellular RNase as quickly as possible in the initial stage of nucleic acid isolation and purification. Under the synergistic effect of β-mercapto Z. alcohol, high concentration (iso)guanidine thiocyanate can inhibit the activity of RNase extremely and quickly, and can isolate complete RNA molecules from pancreas and other RNase-rich tissue cells , Has now become a routinely used reagent. For samples with lower content, adding glycogen can increase the recovery rate of RNA. The one-step method is most commonly used in total RNA extraction.

 

1,(Iso) Guanidine Thiocyanate and Chloroform One Step Method

The (iso)guanidine thiocyanate-phenol chloroform method is a classic one-step method, proposed by Chomczynski and Sacchi in 1987. It lyses the cells with a denaturing solution containing 4mmol/L (iso)guanidine thiocyanate and 0.1mmol/L β-mercaptoethanol, and then extracts the lysis solution with phenol/chloroform under acidic conditions of pH 4.0, and finally RNA is prepared by precipitation with isopropanol and washing with 75% ethanol. Compared with the previous (iso)guanidine thiocyanate-CsCl ultracentrifugation method, this method is simple, economical and efficient, and can handle multiple at the same time. Specimens, and the integrity and purity of RNA are high. It is still used for the isolation and purification of nucleic acids from cultured cells and most animal tissues.

The yield of total RNA depends on the initial amount of the specimen. The yield of total RNA per milligram of tissue is about 4-7 g, and about 5-10 g per 106 cells. However, this method is not suitable for extracting RNA from adipose tissue rich in triglycerides, and sometimes the RNA will be contaminated with polysaccharides and proteoglycans. These contaminations will affect the dissolution of RNA after ethanol precipitation, while inhibiting the RT-PCR reaction. It affects the blotting step in RNA hybridization by binding to the membrane. The extraction of adipose tissue RNA can be replaced by (iso)guanidine thiocyanate-CsC1 ultracentrifugation method. When the pollution of polysaccharides and proteoglycans is serious, the next method can be eliminated by adding an organic solvent extraction step and changing the RNA precipitation conditions.

Scientific Research

2,One-step method that can prepare RNA, DNA and protein at the same time

This method is an improved method of (iso)guanidine thiocyanate-phenol chloroform one-step method. It uses (iso)guanidine thiocyanate-phenol single-phase lysis reagent to lyse cells, and then chloroform is added to form two phases. The denatured DNA and protein are located at the interface of the two phases, and the RNA retained in the upper aqueous phase is prepared by isopropanol precipitation and 75% Z alcohol washing in the RNA precipitation solution. The composition of the RNA precipitation solution is 1.2mmol/L NaCl and 0.8mmol/L disodium citrate.

Due to the use of RNA precipitation solution, the RNA samples prepared by this method are rarely contaminated with polysaccharides and proteoglycans, and can be used for mRNA purification, Northern hybridization, reverse transcription and RT-PCR reactions. The DNA and protein at the interface can be precipitated separately by ethanol and isopropanol. The DNA prepared by this method is about 20 kb in size and can be used as a template for PCR reactions, while protein samples are mainly used for western blotting. At present, this method has a variety of commercially available single-phase lysis reagents to choose from. It is the most commonly used method for total RNA extraction, and its yield is equivalent to (iso)guanidine thiocyanate-phenol chloroform one-step method.

 

3, other methods

Isolation and purification of nucleic acids are numerous, such as LiC1-urea method, (iso)guanidine thiocyanate-CsCl ultracentrifugation method, thermal phenol method, guanidine hydrochloride-organic solvent method, etc., which are now rarely used due to various reasons.

At present, Ascend has formed a professional nucleic acid automatic purification program covering a variety of biological samples.

Classification of nucleic acid extraction magnetic beads

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With the outbreak of the new coronavirus, although it has been brought under control, everyone still cannot take it lightly. Everyone should know that all suspected cases and patients with fever must undergo nucleic acid testing to confirm whether they are new coronaviruses. The steps of nucleic acid detection are mainly divided into three steps:
1. Sampling: Collect samples that may contain viruses from the respiratory tract or other parts of the human body.
2. Extraction: extract the viral nucleic acid from the sample and separate it from other substances.
3. Detection: Detect the extracted virus content by fluorescence quantitative PCR method.
Among them, magnetic beads are one of the necessary tools for nucleic acid extraction. So what are the classification and functions of magnetic beads for nucleic acid extraction?
Classification and function of magnetic beads: According to the surface properties, they can be divided into the following three categories

1. Silicone hydroxy magnetic beads

This is one of the most widely used magnetic beads. When using silanol magnetic beads to extract nucleic acids, high concentrations of chaotropic salts (NaI, NaClO4, GuHCI, GuSCN, etc.) are usually required. High concentration of salt ions can effectively shield the electrostatic repulsion in the solution. At the same time, chaotropic salt ions can also competitively combine with the surface of magnetic beads and nucleic acid molecules, destroy the original hydration layer on both surfaces, and promote the adsorption (hydrogen bond + van der Waals force) between the surface of magnetic beads and nucleic acid molecules. Since commonly used chaotropic salts such as guanidine ions have an inhibitory effect on PCR, they must be removed in the subsequent washing steps. The removal effect can be monitored by the absorbance ratio.


2. Carboxy magnetic beads

In addition to being able to bind nucleic acid molecules in a high-concentration chaotropic salt environment, such magnetic beads can bind nucleic acid molecules through another special mechanism. By adding a certain concentration of PEG and NaCl to the solution, the nucleic acid molecules can gradually curl up from the stretched conformation to a small ball shape, and most of the negative charges on it are also shielded, which promotes the adsorption of the nucleic acid molecules to the magnetic beads. The larger the molecular weight of the nucleic acid molecule, the more likely it is that this conformational change from the stretched coil to the curled down ball will occur. Therefore, by adjusting the volume ratio of the salt solution to the nucleic acid sample, it is possible to achieve a larger molecular weight nucleic acid fragment on the carboxyl magnetic beads. The preferential adsorption on the surface achieves the so-called fragment screening effect.

3. Amino/imidazolyl and other positively charged magnetic beads

Such magnetic beads can easily realize the reversible binding of nucleic acid molecules on the surface by adjusting the pH. However, because the positively charged surface adsorbs nucleic acid molecules too strongly, it is easy to cause the yield to be low. Therefore, the use of positively charged magnetic beads is not as widespread as silanol and carboxyl magnetic beads.
The classification and function of magnetic beads for nucleic acid extraction are explained in detail above. Aisen uses nano-biomagnetic beads as the core to produce nucleic acid extractors and their matching magnetic beads nucleic acid extraction kits.

What are the principles of nucleic acid extraction and purification?

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Nucleic acid extraction is used in molecular biochemical tests and diagnosis, and is the first step of cloning, transformation, enzyme digestion, in vitro transcription, amplification, and sequencing. However, due to the large amount of proteins, carbohydrates, metabolites and other contaminants in the original sample, it is not easy to extract high-quality nucleic acids. It is necessary to apply the supporting nucleic acid extraction system and nucleic acid extraction reagents to automatically complete the extraction of sample nucleic acid.

Principles of nucleic acid extraction and purification

1. The integrity of the primary structure of nucleic acid should be guaranteed;
2. Eliminate pollution from other molecules.
In order to ensure the study of nucleic acid structure and function, a complete primary structure is the most basic requirement, because genetic information is all stored in the primary structure, and the primary structure of nucleic acid also determines the form of its high-level structure and other biological macromolecules. The way of combining. The purification of nucleic acid should meet the following three requirements:
1. There are no organic solvents and excessively high concentrations of metal ions that can inhibit enzymes in nucleic acid samples;
2. The pollution of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized;
3. Eliminate the contamination of other nucleic acid molecules, such as RNA should be removed when extracting DNA molecules, and vice versa.


In order to ensure the integrity and purity of the isolated nucleic acid, the following items should be noted during the experiment:
1. Try to simplify the operation steps and shorten the extraction process to reduce the damage of various harmful factors to the nucleic acid;
2. Reduce the degradation of nucleic acids by chemical factors. In order to avoid damage to the phosphodiester bond in the nucleic acid chain by over acid and over base, the operation is mostly carried out under the condition of pH 4-10;
3. Reduce the degradation of nucleic acids by physical factors. The main physical degradation factor is mechanical shearing force, followed by high temperature.
Mechanical shearing force includes strong and high-speed solution shaking and stirring to make the solution quickly pass through the narrow and long pores, the cells are suddenly placed in the hypotonic solution, the cells burst explosively, and the DNA samples are repeatedly frozen and stored. These operating details should be paid attention to in the experimental operation. The main hazard of mechanical shearing is linear DNA molecules with large molecular weights, such as the chromosomal DNA of eukaryotic cells.
The threat to circular DNA molecules with small molecular weights, such as plasmid DNA and RNA molecules, is relatively small. High temperature is like boiling for a long time. In addition to the shear force caused by water boiling, the high temperature itself can also damage some chemical bonds in nucleic acid molecules. In the process of nucleic acid extraction, it is generally operated at low temperature, but now it is found that there is not much difference in the quality of nucleic acid obtained at room temperature rapid extraction and low temperature extraction.
4. To prevent the biodegradation of nucleic acids, various nucleases inside or outside the cell digest the phosphodiester bond in the nucleic acid chain and directly destroy the primary structure of the nucleic acid. Among them, DNA enzymes require the activation of metal divalent ions Mg2 and Ca2. Using EDTA and citrate to chelate metal divalent ions can basically inhibit DNase activity. RNase is not only widely distributed, easily contaminates the sample, but also resistant to high temperature, acid, alkali, and not easy to inactivate, so it is the main hazard factor in the biodegradable RNA extraction process.
The main steps of nucleic acid extraction are nothing more than disrupting cells, removing proteins bound to nucleic acids, polysaccharides, lipids and other biological macromolecules, removing other unwanted nucleic acid molecules, precipitating nucleic acids, removing salts, organic solvents and other impurities, and purifying nucleic acids Wait. The nucleic acid extraction scheme should be determined according to the characteristics of the specific biological material and the nucleic acid molecules to be extracted. For the nucleic acid molecules enriched in a specific organelle, the scheme of extracting the organelle in advance and then extracting the target nucleic acid molecule can obtain completeness and Nucleic acid molecules with high purity and high quality.

Use method and precautions of magnetic bead method nucleic acid extraction system

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The nucleic acid extraction system is an instrument that uses matching nucleic acid extraction reagents to automatically complete sample nucleic acid extraction. The magnetic bead method is to lyse the cell tissue sample through the lysis solution, the nucleic acid molecules free from the sample are specifically adsorbed to the surface of the magnetic particles, and the impurities such as proteins are not adsorbed but remain in the solution. The magnetic beads carrying nucleic acid are adsorbed by a magnetic rod and moved to different reagent tanks. The pure nucleic acid is finally obtained by repeated rapid stirring and mixing of the liquid, cell lysis, nucleic acid adsorption, washing and elution and other steps.

Use of magnetic bead nucleic acid extraction system

There are usually two ways to use the magnetic bead method nucleic acid extraction system, namely the magnetic rod method and the suction method. Below we will explain the specific operations of the magnetic rod method and the suction method in detail.

1. Magnetic rod method

The magnetic rod method uses the transfer of magnetic beads and the immobilization of liquids to realize the separation of nucleic acids. The principle and process are the same as the suction method. There is a difference in the separation between magnetic beads and liquid. The magnetic rod method is to separate the magnetic beads from the waste liquid through the adsorption of the magnetic beads on the magnetic rod, and put them in the liquid in the next step to realize the extraction of nucleic acid.

2. Suction method

The suction method is also called the pipetting method, which uses the transfer of liquid or the immobilization of magnetic beads to realize the extraction of nucleic acid. The transfer is usually realized by the operation of the system control manipulator, and the extraction process is as follows:

1) Cracking

Add the lysate to the sample, use mechanical movement and heating to mix the reaction solution and make the reaction complete. Lyse the cell to release the nucleic acid.

2) Adsorption

Add the magnetic beads to the sample lysing solution, mix well, and use the magnetic beads to have a strong affinity for nucleic acids under high salt and low pH values. The magnetic beads can adsorb nucleic acids. Under the action of an external magnetic field, separate the magnetic beads from the solution. Transfer the tip to the waste tank and discard the tip.

3) Washing

Remove the external magnetic field, use a new tip to add the washing buffer, mix well, remove impurities, and remove the liquid under the action of the external magnetic field.

4) Elution

Remove the external magnetic field, add the elution buffer with a new tip, mix thoroughly, and the bound nucleic acid is separated from the magnetic beads, so that the purified nucleic acid is obtained.

HERO-96

Precautions

1. The nucleic acid extraction system is at least 10cm away from other vertical surfaces.

2. The input power cord of the instrument must be grounded to prevent electric shock accidents.

3. The operator is not allowed to disassemble the instrument without authorization, and must have a qualified professional maintenance personnel to complete the replacement of components or perform internal adjustments and other operations. When the power is turned on, do not replace the components.

4. The relative humidity is 10%-80%, the unobstructed air is 35℃ or below, the normal atmospheric pressure (altitude should be lower than 3000m), the temperature 20-35℃, and the typical use temperature 25℃ is the installation environment of the instrument.

5. Do not place the instrument near a heat source such as an electric heater. Avoid splashing water or other liquids into the electronic components to avoid short circuits.

6. Both the air inlet and the air outlet are located at the back of the instrument, and at the same time, prevent dust or fibers from accumulating at the air inlet and keep the air duct unobstructed.

At present, the magnetic bead method nucleic acid extractor is widely used in various fields such as environmental microbial detection, food safety detection, blood transfusion safety, forensic medicine identification, disease control center, clinical disease diagnosis, biological research, and animal husbandry.

What are biomagnetic beads and how to buy them?

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The so-called biomagnetic beads refer to superparamagnetic microspheres with fine particle size. Magnetic beads generally have super-strong paramagnetism and can quickly gather in a magnetic field, and after leaving the magnetic field, they can help to uniformly disperse the magnetic separation. Secondly, it has a suitable particle size with small difference to ensure a strong enough magnetic response without sedimentation. Thirdly, it has abundant surface-active groups so that it can be coupled with biochemical substances and separated from the sample to be tested under the action of an external magnetic field.

Compared with traditional separation methods, the use of magnetic beads for the separation of complex components of biochemical samples can achieve separation and enrichment at the same time, which effectively improves the separation speed and enrichment efficiency, and also makes the analysis and detection sensitivity more sensitive. Huge improvements.

Characteristics of biological magnetic beads in nucleic acid extraction

(1) Biological magnetic beads can realize automation and mass operation, meet the high-throughput operation requirements of biology, and facilitate rapid and timely response to major disease outbreaks.

(2) Simple operation and short time. The entire extraction process has only four steps, most of which can be completed within 36-60 minutes;

(3) It is safe and non-toxic, does not use any toxic reagents, and conforms to modern environmental protection concepts; it provides a healthy environment for the bodies of our scientific researchers and enhances our research confidence;

(4) The specific combination of magnetic beads and nucleic acid makes the extracted nucleic acid purity and concentration high. Provided a guarantee for our researchers’ follow-up experiments.

Amino Magnetic Beads

Application field of biomagnetic beads

(1) Nucleic acid extraction, magnetic bead DNA extraction is a perfect combination of nanotechnology and biotechnology, and has advantages that traditional DNA extraction methods cannot match. Nucleic acid extraction is the basis of the biomedical field. With its high efficiency and stability, magnetic bead DNA extraction is of great significance to the rapid development of genetic testing and personalized medicine.

(2) Cell separation (CD4, CD8) can be further used for tumor detection and diagnosis.

(3) Protein and antibody separation (amino magnetic beads, carboxyl magnetic beads, epoxy magnetic beads), which can be further used for disease detection and diagnosis.

(4) Heavy metal detection (sulfhydryl magnetic beads), which can be further used for sewage treatment, etc.

(5) Nuclear magnetic resonance (non-coated magnetic beads) is used for contrast agents.

At the same time, biological magnetic beads (or biological microspheres), as an effective new carrier for nucleic acid extraction, have been recognized by the majority of scientific researchers. The industry is referred to as “magnetic bead method”. Work efficiency has gradually become the mainstream solution.

However, the quality of the magnetic beads varies from good to bad, and the extraction effect is not satisfactory, which is really troublesome, and even doubts the feasibility of this new extraction scheme. So, how should we choose a biomagnetic bead?

1. Settling speed

Settling speed is often an important indicator. The slower the settling speed, the better the uniformity of sample addition, and the better the operating experience. However, considering the comprehensive performance of the magnetic beads, the settling speed is generally controlled at 10-20min, and some fast magnetic beads , The sedimentation speed is only 3-5min, which requires attention to the mixing frequency of the magnetic beads in the actual operation, so as to avoid uneven sample addition and unstable results.

Carboxyl Magnetic Beads

2. Magnetic time

The short magnetic attraction time indicates that the magnetic beads are more responsive and easier in actual operation. However, the shorter the magnetic attraction time, the faster the settling speed. Generally speaking, the magnetic attraction time is within the acceptable range within 30s. However, some brands of magnetic beads, including imported magnetic beads, have a magnetic attraction time of more than 1 min. This will significantly increase the waiting time during the operation and reduce the operation efficiency.

3. Nucleic acid adsorption capacity

Many customers believe that the nucleic acid adsorption capacity of magnetic beads is related to the particle size of the magnetic beads. Magnetic beads with a small particle size have a large specific surface area, so the adsorption capacity is strong. This conclusion is theoretically valid, but in actual operation, when the magnetic beads are used for nucleic acid adsorption, 5-6 kinds of reagents are needed. Therefore, we want to make the magnetic beads absorb more nucleic acids. The entire reagent system and the magnetic beads The compatibility of reagents is an important factor affecting the extraction results. Magnetic beads with a small particle size are not suitable for any reagent system without optimization. Therefore, instead of spending energy to find magnetic beads with a smaller particle size, it is better to optimize the entire reagent system. The efficiency may be higher.

Whether it is a scientific research user or an industrial user, choosing a standard magnetic bead manufacturer is the key to ensuring the stable progress of the project. Biomagnetic beads are a cross product of synthetic chemistry and molecular biology, and their applications are concentrated in the field of molecular biology. From this perspective, biological companies will solve more practical applications.

Therefore, for users, the docking of magnetic beads products with biological companies will be more efficient in terms of after-sales service. It is recommended that you look for standard and biological magnetic beads during the process of purchasing magnetic beads. Quotient.

What are the nucleic acid extraction methods?

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Nucleic acid is the carrier of genetic information, the most important biological information molecule, and the main object of molecular biology research. Therefore, nucleic acid extraction is the most important and basic operation in molecular biology experimental technology.

Nucleic acid is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). RNA can be divided into ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA) according to their functions.

DNA is mainly concentrated in the nucleus, mitochondria and chloroplasts, while RNA is mainly distributed in the cytoplasm.

In nucleic acids, purine bases and pyrimidine bases have conjugated double bonds, so nucleic acids have ultraviolet absorption characteristics. The ultraviolet absorption of DNA sodium salt is around 260nm, and its absorbance is represented by A260. It is in the absorption trough at 230nm, so ultraviolet spectroscopy can be used. The photometer performs quantitative and qualitative determination of nucleic acids.

Nucleic acid is an amphoteric electrolyte, which is equivalent to a polyacid. A neutral or alkaline buffer can be used to dissociate nucleic acid into anions, which are placed in an electric field and move towards the anode. This is the principle of electrophoresis.

Principles and requirements of nucleic acid extraction and purification

1. Ensure the integrity of the primary structure of nucleic acid;

2. Eliminate pollution from other molecules (such as eliminating RNA interference when extracting DNA);

3. Nucleic acid extraction kit samples should not contain organic solvents that can inhibit enzymes and high concentrations of metal ions;

4. Minimize macromolecular substances such as proteins, polysaccharides and lipids as much as possible.

Nucleic acid extraction type

products

1. Extraction of total RNA

Of the total RNA, 75-85% is rRNA (mainly 28S-26S/23S and 18S/16S rRNA), and the rest consists of mRNA and small RNA with different molecular weights and nucleotide sequences such as tRNA, 5S rRNA, 5.8S rRNA, miRNA, siRNA, small nuclear RNA (small nuclear RNA, snRNA) and small nucleolar RNA (small nuceolar RNA, snoRNA) and other components.

2. miRNA extraction

MicroRNAs (miRNAs) are small, highly conserved RNA molecules, such as small interfering RNAs (siRNAs), which regulate the expression of their homologous mRNA molecules by base pairing with them to prevent expression through various mechanisms. They have become a key regulatory agency for development, cell proliferation, differentiation and cell cycle.

3. Genomic DNA extraction

For gene structure and function research and gene diagnosis, it is usually required that the length of the obtained fragment is not less than 100-200kb. In the DNA extraction process, various factors that cause DNA fragmentation and degradation should be avoided as much as possible to ensure the integrity of DNA and lay the foundation for subsequent experiments.

4. Plasmid extraction

The plasmid extraction method is to remove RNA, separate the plasmid from the bacterial genomic DNA, and remove proteins and other impurities to obtain a relatively pure plasmid.

Nucleic acid extraction and purification method

Pre-packed nucleic acid extraction kit

1. Phenol/chloroform extraction method

Invented in 1956, after phenol/chloroform treatment of cell crushing liquid or tissue homogenate, nucleic acid components mainly composed of DNA are dissolved in the aqueous phase, and lipids are mainly contained in the organic phase, and proteins are located between the two phases.

2. Alcohol precipitation method

Ethanol can eliminate the hydration layer of nucleic acid and expose the negatively charged phosphate groups. Positively charged ions such as NA﹢ can combine with the phosphate groups to form a precipitate.

3. Chromatography column method

Through the special silicon matrix adsorption material, DNA can be specifically adsorbed, and RNA and protein can pass through smoothly, and then use high salt and low pH to bind nucleic acid, and low salt and high pH elution to separate and purify nucleic acid.

4. Thermal cracking alkali method

Alkaline extraction mainly uses the topological difference between covalently closed circular plasmids and linear chromatin to separate them. Under alkaline conditions, denatured proteins are soluble.

5. Boiling cracking method

Heat the DNA solution to use the characteristics of linear DNA molecules to separate the DNA fragments from the precipitate formed by denatured proteins and cell debris by centrifugation.

6. Nano magnetic bead method

The surface of superparamagnetic nanoparticles is modified and modified by nanotechnology to prepare superparamagnetic silicon oxide nanomagnetic beads. The magnetic beads can specifically recognize and efficiently combine with nucleic acid molecules on the microscopic interface. Using the superparamagnetism of silica nanospheres, under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate, etc.) and an external magnetic field, DNA and RNA are separated from blood, animal tissues, food, pathogenic microorganisms and other samples .

7. Other methods

In addition to the above-mentioned commonly used methods, there are multiple methods such as ultrasound, repeated freezing and thawing, enzymatic hydrolysis, and hypotonic lysis.

To stop the new coronavirus (2019-nCoV), Aisen is in action

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As of 0:00 on January 23, a total of 571 cases of novel coronavirus pneumonia have been confirmed in my country, and 17 cases have died.

Changes in the pneumonia epidemic caused by new coronavirus infection in Wuhan

Date

Cumulative confirmed cases

Cumulative cured and discharged

Treating severe illness

Cumulative deaths

1.14

41
7

6

1

1.15

41

12

5

2

1.16

45

15

5

2

1.17

62

19

8

2

1.18

121

24

——

3
1.19
198

25

44

3

As a professional manufacturer of nucleic acid extraction reagents, Ascend Biotech adjusted the production arrangement at the first time, and prepared nucleic acid extraction reagents (magnetic bead method) and virus detection reagents that can be automated.

Product advantages:

1. Automated detection. With the HERO 32 nucleic acid extraction system independently developed by Aisen Biotechnology, you only need to add the sample to the deep-well plate and run the relevant program. Multiple samples can be extracted within 15-60 minutes, which is efficient and fast, and can protect the experimental operators. Security.

2. Large sample size for single test. The kit has two sizes of 64T and 50T, which can extract multiple samples in one extraction, reducing waiting time and increasing efficiency.

3. Specificity and accuracy. The special magnetic bead extraction principle makes the kit have high specificity and accuracy for RNA extraction.

Luoyang Ascend Biotechnology Co., Ltd. is a professional manufacturer of nucleic acid extraction reagents focusing on the sample processing field of life sciences. Aisen takes nano-biomagnetic beads as the core to produce nucleic acid extraction instrument and its supporting magnetic bead method nucleic acid extraction kit. You only need to provide design solutions according to your needs.

Ascend will provide you with strong production capacity and strict quality control. Service, be your strong market backing. In 2019, in the prevention and control of the epidemic, Essen has provided nearly one million reagents, which has effectively contained the spread of the epidemic and blocked the new coronavirus (2019-nCoV). Essen is still fighting!

Research shows: genetic abnormalities are related to cerebral palsy

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A new study published in the journal Nature found that there is an association between cerebral palsy and genetic variation. This surprising discovery challenges the claim that environmental factors are the only cause of cerebral palsy.

According to study co-author Stephen Scherer, many people were not optimistic about this study at first, because for many years it was believed that genes had no effect on cerebral palsy.

After examining the genes of 115 children with cerebral palsy and their parents, the researchers found that 10% of these children had chromosomal abnormalities. Scherer said that 10% is a very significant ratio, and this discovery is very shocking.

According to the researchers, the location of the problem with the gene of the child with the mutation is in the copy number variation region.

It is understood that copy number variation refers to the increase or decrease in the copy number of large genome fragments with a length of more than 1 kb. The variation will cause only one or three copies of the gene copied from both parents. One of the genes may be Deleted or copied additionally.

Scherer said that everyone has copy number variation, but the children in the study have a lot of copy number variation, which affects dozens to hundreds of genes. Before this, scientists have always believed that cerebral palsy, a common disease that causes physical disabilities in children, is caused by infections or strokes that occur in babies during pregnancy or at birth. People will consider genetic factors only when there is no obvious environmental impact. .

At present, researchers believe that genetic testing should also become a standard testing method to help parents detect abnormalities in their children and detect other diseases early.

BGI’s valuation dispute: 19 daily limit market value of 38 billion yuan, with 1.6 billion assets

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A reporter from the Beijing News noted that BGI has raised funds several times in the past two years, with more than 40 institutional shareholders behind it.

The prospectus shows that before going public, BGI has mainly experienced three rounds of financing: in 2012 for BGI Technology, BGI Medical in 2014, and 2015 after the medical and technology reorganization of BGI shares.

In 2012, Huada Holdings announced the acquisition of Complete Genomics, a US gene sequencing company. In order to raise funds, Huada sold 42% of its subsidiary Huada Technology and raised 1.398 billion yuan. It was led by Sequoia Capital and China Everbright, Shenzhen Venture Capital , Yunfeng Investment, Jinglin Assets, Taishan Investment, Softbank China, Shengqiao Investment and other well-known institutions are shortlisted for financing.

In May 2014, BGI began to introduce external institutional investors. According to the overall valuation of 10 billion, 8 external institutional investors increased the registered capital of BGI by a total of 295 million yuan, which accounted for 2.0824 million yuan of registered capital after the capital increase. 3.35%.

In the first half of 2015, Huada Medical once again introduced external investment institutions such as Heyu Gaolin and China Life Insurance, and based on the overall valuation of approximately 19.1 billion yuan in the total equity value of Huada Medical and Huada Technology before the investment as the capital increase and transfer Pricing basis. Among them, Shenzhen Heyu Gaolin acquired 35,849,600 shares of Huada Medical with a capital investment of 2 billion yuan, becoming the third largest shareholder after Huada Holdings and Huada Investment.

Statistics show that after three rounds of financing, BGI, with BGI Medical and BGI Technology as the main body, introduced more than 40 institutional investors, with a total investment of 7.215 billion yuan, while BGI has cashed out 4.296 billion. yuan.

The pre-IPO valuation of Huada Gene was as high as 19 billion yuan. After experiencing a lower limit on August 11, the market value of Huada Gene was around 38 billion yuan. In the last round of shares in Huada Gene, the book value was only about double. Taking Rongzhilian as an example, it once disclosed that the cost of investing in Huada Gene was 35 million yuan. Today, the market value of BGI’s shares held by Rongzhilian is 89.714 million yuan. However, the prospectus shows that shareholders of Huada Genetics Institution will be able to lift the ban after one year.

Regarding the market value of Huada Gene, the valuation given by securities firms is not high. The Beijing News reporter noted that since the IPO of Huada Gene, more than 14 brokerage agencies have issued research reports on it. The highest stock price of the brokerage institution for Huada Gene is 94.4 yuan per share, and the lowest is 46.35 yuan per share.

In the latest research report, Caitong Securities analyzed that Huada Gene’s reasonable market value was 25.83 billion-34.44 billion yuan, corresponding to a reasonable range of 64.57-86.09 yuan in 2017. Southwest Securities gave BGI a P/E ratio of 60-80 times in 2017, corresponding to a reasonable market value range of 25.8 billion-34.4 billion yuan, and a stock price range of 70.8-94.4 yuan.

Zero2IPO’s return on IPOs with institutional support for listing in the first quarter of this year shows that Shenzhen’s GEM listed companies had an average book investment return of 2.42 times on the date of issue, and after 20 trading days of listing, the average book return of institutions was 9.44. Times. BGI now happens to be listed on the 20th day of trading. Calculated at a rate of return of 9.44 times, the market value should be 100 billion yuan, much higher than the current market value of BGI.

Reproductive health services account for more than half of revenue

According to the prospectus, from 2014 to 2016, BGI’s operating income was 1.132 billion yuan, 1.319 billion yuan and 1.711 billion yuan, and net profits were 59 million yuan, 272 million yuan and 350 million yuan respectively. Among them, reproductive health services have been BGI’s main source of revenue in recent years, accounting for an increase from 31.71% in 2014 to 54.62% in 2016.

In 2016, reproductive health services brought BGI Gene’s revenue of 929 million yuan, with a gross profit margin of 76.41%. BGI’s prospectus shows that the service items provided by reproductive health services mainly include non-invasive fetal chromosomal abnormality detection, neonatal deafness gene detection, neonatal genetic metabolic disease screening, and single gene disease detection. Among them, non-invasive prenatal screening (NIPT) to prevent neonatal defects is the most mature field of clinical application of gene sequencing.

BGI did not disclose its NIPT market share in the prospectus, but the backdoor announcements of its peers, Berry and Kang, can compare the situation of BGI. Berry and Kang, founded in 2010, also originated from BGI. The reorganization plan of Berry and Kang Backdoor *ST Tianyi shows that its founder and chairman Gao Yang previously served as the general manager of the BGI Gene Health Division.

Berry and Kang disclosed in the backdoor restructuring plan that in 2016, it achieved revenue of 922 million yuan. Berry Hekang’s business is almost all non-invasive prenatal genetic testing and instrument kit sales, similar to BGI’s reproductive health genetic testing services. In 2016, the reproductive health services brought BGI’s revenue of 929 million yuan. .

Berry Hekang disclosed in the backdoor restructuring plan that in 2016, Berry Hekang’s market share in the domestic NIPT service sector was approximately 33.06%-37.19%. BGI’s revenue in the NIPT service field is similar to that of Berry and Kang, and their market share should be similar.

The NIPT industry report shows that my country’s NIPT market was basically monopolized by BGI and Berry and Kang in the early stage. In 2013, BGI and Berry and Kang accounted for 46%, and Berry and Kang accounted for 40%. Looking at the latest market share of BGI, Berry and Kang, BGI has declined significantly. The report said that in the field of genetic testing services for reproductive health, where the market is gradually stabilizing, competition among companies will become more intense.

Has 1.6 billion yuan of wealth management products before listing

Compared with other companies that choose to go public for financing to obtain corporate development funds, BGI can be described as “not bad money”. The prospectus shows that the debt ratio of BGI in 2014 was only 28.47%, and the debt ratio has continued to decline in the following two years. By the end of 2016, BGI’s total debt was 778 million yuan, and the total assets of the same period were 4.23 billion yuan, and the debt ratio was only 18.38%. .

In the breakdown of BGI’s liabilities, its main liabilities are operating liabilities, among which the amount of advance accounts received over the years is relatively large, and the proportion of liabilities exceeds 50%. In 2016, BGI Gene received 438 million yuan in advance, accounting for 56.3% of total liabilities.

Financial liabilities account for very little in the balance sheet of BGI. From 2014 to 2016, BGI Gene’s short-term loans over the years were all in the millions. At the end of 2016, BGI’s short-term loan was only 3 million yuan, and there was no long-term loan.

With little financial liabilities, BGI’s assets have grown rapidly in the past two years. In 2014, BGI Gene’s total assets were 2.05 billion yuan. At the end of 2016, the total assets data increased to 4.23 billion yuan, and the total assets doubled in two years.

The increase in BGI’s assets is mainly reflected in other current assets. In 2014, BGI’s other current assets were only 12.5142 million yuan, which increased to 1.883 billion yuan in 2015. Although it decreased in 2016, it was still as high as 1.726 billion yuan. Yuan, accounting for 53.89% of BGI’s current assets.

BGI’s prospectus shows that in its billions of other current assets, bank wealth management products account for almost all. For example, among other current assets of 1.883 billion yuan in 2015, the balance of wealth management products and interest was 1.86 billion yuan, accounting for 98.79%.

As of the end of 2016, the related wealth management products and interest sold by large commercial banks such as Bank of China, Industrial and Commercial Bank of China, and Agricultural Bank of BGI totaled 1.609 billion yuan. That year, BGI’s interest income increased to 73,465,800 yuan.

The funds used by BGI to purchase wealth management may be derived from its financing income in recent years. The prospectus shows that in 2015, the net cash activity generated by BGI’s financing activities was 1.651 billion yuan. At the end of 2015, the balance of BGI’s monetary funds increased by less than 8 million yuan compared with 2014. In the same period, BGI’s The net outflow of foreign investment was 1.978 billion yuan.

The debt ratio is low, and most of the more than one billion yuan in foreign financing is used to purchase bank financing, which shows that BGI is “not bad for money.” The prospectus shows that BGI’s IPO raised an amount of 547 million yuan and the net amount of funds raised was 483 million yuan, which is only equivalent to 30% of BGI’s financial management funds at the end of 2016.

Most of the funds raised by the IPO are still used by BGI to purchase wealth management products. On August 11, BGI announced that it decided to use temporarily idle raised funds with a quota of no more than 300 million yuan for cash management and purchase high-security bank-guaranteed wealth management products.

The current stock price exceeds the target price given by the broker

A reporter from the Beijing News noted that BGI has raised funds several times in the past two years, with more than 40 institutional shareholders behind it.

The prospectus shows that before going public, BGI has mainly experienced three rounds of financing: in 2012 for BGI Technology, BGI Medical in 2014, and 2015 after the medical and technology reorganization of BGI shares.

In 2012, Huada Holdings announced the acquisition of Complete Genomics, a US gene sequencing company. In order to raise funds, Huada sold 42% of its subsidiary Huada Technology and raised 1.398 billion yuan. It was led by Sequoia Capital and China Everbright, Shenzhen Venture Capital , Yunfeng Investment, Jinglin Assets, Taishan Investment, Softbank China, Shengqiao Investment and other well-known institutions are shortlisted for financing.

In May 2014, BGI began to introduce external institutional investors. According to the overall valuation of 10 billion, 8 external institutional investors increased the registered capital of BGI by a total of 295 million yuan, which accounted for 2.0824 million yuan of registered capital after the capital increase. 3.35%.

In the first half of 2015, Huada Medical once again introduced external investment institutions such as Heyu Gaolin and China Life Insurance, and based on the overall valuation of approximately 19.1 billion yuan in the total equity value of Huada Medical and Huada Technology before the investment as the capital increase and transfer Pricing basis. Among them, Shenzhen Heyu Gaolin acquired 35,849,600 shares of Huada Medical with a capital investment of 2 billion yuan, becoming the third largest shareholder after Huada Holdings and Huada Investment.

Statistics show that after three rounds of financing, BGI, with BGI Medical and BGI Technology as the main body, introduced more than 40 institutional investors, with a total investment of 7.215 billion yuan, while BGI has cashed out 4.296 billion. yuan.

The pre-IPO valuation of Huada Gene was as high as 19 billion yuan. After experiencing a lower limit on August 11, the market value of Huada Gene was around 38 billion yuan. In the last round of shares in Huada Gene, the book value was only about double. Taking Rongzhilian as an example, it once disclosed that the cost of investing in Huada Gene was 35 million yuan. Today, the market value of BGI’s shares held by Rongzhilian is 89.714 million yuan. However, the prospectus shows that shareholders of Huada Genetics Institution will be able to lift the ban after one year.

Regarding the market value of Huada Gene, the valuation given by securities firms is not high. The Beijing News reporter noted that since the IPO of Huada Gene, more than 14 brokerage agencies have issued research reports on it. The highest stock price of the brokerage institution for Huada Gene is 94.4 yuan per share, and the lowest is 46.35 yuan per share.

In the latest research report, Caitong Securities analyzed that Huada Gene’s reasonable market value was 25.83 billion-34.44 billion yuan, corresponding to a reasonable range of 64.57-86.09 yuan in 2017. Southwest Securities gave BGI a P/E ratio of 60-80 times in 2017, corresponding to a reasonable market value range of 25.8 billion-34.4 billion yuan, and a stock price range of 70.8-94.4 yuan.

Zero2IPO’s return on IPOs with institutional support for listing in the first quarter of this year shows that Shenzhen’s GEM listed companies had an average book investment return of 2.42 times on the date of issue, and after 20 trading days of listing, the average book return of institutions was 9.44. Times. BGI now happens to be listed on the 20th day of trading. Calculated at a rate of return of 9.44 times, the market value should be 100 billion yuan, much higher than the current market value of BGI.