August 16, 2022

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Nucleic Acid Knowledge Series (1)

Nucleic Acid Knowledge Series (1)



 

Nucleic Acid Knowledge Series (1). 

Nucleic acid extraction includes two major steps: lysis and purification of the sample. Lysis is the process of freeing the nucleic acid in the sample in the lysis system.

Purification is the process of completely separating the nucleic acid from other components in the lysis system, such as proteins, salts and other impurities.

 

Nucleic Acid Knowledge Series (1)

 

 

Almost all classic lysates contain detergents (such as SDS, Tween 20, etc.) and salts (such as Tris, EDTA, NaCl, etc.).

 

The main function of detergents is to achieve the release of nucleic acids in the lysis system by denaturing the protein, destroying the membrane structure and unraveling the protein connected to the nucleic acid.

Protease is generally added to the lysis system, and the protein is digested into small fragments by protease to promote the separation of nucleic acid and protein.

At the same time, it is also convenient for subsequent purification operations and obtain more pure nucleic acid. There are also direct use of high-concentration protein denaturants (such as GIT, GuHCl, etc.) for lysis.

This method has become the mainstream of RNA extraction, but not the mainstream of genomic DNA extraction.

In addition to providing a suitable lysis environment (such as Tris), the main role of salt also includes inhibiting nucleic acid damage (such as EDTA) during the lysis process by nuclease in the sample, and maintaining the stability of nucleic acid structure (such as NaCl).

 

Tips: The biological membrane system of the cell refers to the cell membrane, organelle membrane, and nuclear membrane.

There are ribosomes and centrosomes without membranes; cell membranes, endoplasmic reticulum membranes, Golgi membranes, vacuolar membranes and lysosomal membranes for single membranes; nuclear membranes and mitochondrial membranes (and chloroplast membranes) for double membranes.

 

The most commonly used purification methods are PC extraction + alcohol precipitation, and media purification.

The first method is to use PC to perform repeated extractions on the lysis system to remove protein and realize the separation of nucleic acid and protein; then use alcohol to precipitate nucleic acid to realize the separation of nucleic acid and salt.

The second method is to use certain solid media (magnetic bead purification), under certain specific conditions, to selectively adsorb nucleic acids instead of proteins and salts, to achieve the separation of nucleic acids from proteins and salts.

 

 

 

The role of EDTA when extracting DNA

General blood collection for genetic testing will tell you to use EDTA anticoagulation vacuum blood collection tubes for blood collection, so why use EDTA blood collection tubes (or sodium citrate anticoagulation blood collection tubes)?  It is not recommended to use other blood collection tubes (such as heparin anticoagulation blood collection tubes)?

 

It is generally recommended not to use heparin as an anticoagulant for genetic testing, because heparin not only affects the lysis of blood cells by the lysate and thus affects the extraction of DNA, but also is an inhibitor of the activity of some enzymes (such as Taq enzyme), so heparin-treated When the blood sample is directly subjected to PCR amplification, it will inhibit the activity of Taq enzyme, which will affect the amplification effect (PCR reaction cannot be carried out).

 

Tips: Heparinase I can degrade heparin. If heparinase I is added to the PCR reaction system, it may promote the smooth progress of the PCR reaction.

The general amount of heparin is to add 1ml (0.5U/ml) of heparinase to a 50ml PCR reaction system

 

EDTA (ethylene diamine tetraacetic acid) blood collection tubes generally include EDTA disodium, dipotassium and tripotassium. They can be combined with magnesium ions (Mg2+) and calcium ions (Ca2+) to form a chelate to prevent blood coagulation.

At the same time, they can also inhibit the degradation of DNA by deoxyribonuclease (DNase), because DNase requires a certain amount of metal ions.

As a prosthetic group. At the same time, the presence of EDTA is conducive to the action of lysozyme, because the reaction of lysozyme (with bacteriolytic function) requires an environment with lower ionic strength.

 

 

 

The role of PK enzymes when extracting DNA

Generally proteinase K is not used for plasmid extraction, and proteinase K is generally used for genome extraction.

 

Proteinase K is a serine protease with wide cleavage activity. It cleaves the carboxy terminal peptide bonds of aliphatic amino acids and aromatic amino acids.

This enzyme has been purified to remove RNase and DNase activities.

Because proteinase K is stable in urea and SDS, and has the ability to degrade natural proteins, it has a wide range of applications, including preparation of chromosomal DNA, western blotting, and removal of nucleases in DNA and RNA preparation.

 

The function of proteinase K is to degrade membrane protein, degrade the protein bound to DNA, and make DNA fully free.

Usually, some proteolytic enzymes are used to remove most of the protein before organic solvent extraction. These broad-spectrum proteolytic enzymes include pronase and proteinase K.

 

 

 

Need to add RNase for DNA extraction

The purpose of adding RNase is to obtain pure DNA as much as possible, so it is also possible not to add it.

 

But if RNase is added during DNA extraction, in order to purify DNA, excess RNse must be removed because RNase itself is a protein.

At this time, SDS and KAc (or NaAc) can be added for processing.

Adding SDS can make them become SDS-protein complex precipitates, and then adding KAc to convert these complexes into SDS-protein complexes in the form of less soluble potassium salts.

Make the precipitation more complete. Saturated phenol and chloroform can also be used for extraction and reprecipitation to remove RNase.

 

Tips: SDS, Sodium dodecyl sulfate (SDS), is an anionic detergent commonly used when separating DNA.

It is often used to separate the protein from the DNA after denaturation during the DNA extraction process to dissolve membrane proteins and Fat, which ruptures the cell membrane; dissolves the nuclear membrane and nucleosomes, depolymerizes them, and releases nucleic acids; it has a certain inhibitory effect on RNase and Dnase; SDS can combine with proteins to form R1-O-SO3-…R2+- Protein complexes, denature and precipitate proteins.

 

 

Why use absolute ethanol to precipitate DNA

 

Precipitating DNA with absolute ethanol is the most commonly used method of precipitating DNA in experiments.

The advantage of ethanol is that it can be miscible with water at any ratio. Ethanol will not cause any chemical reaction with nucleic acid and is safe for DNA, so it is an ideal precipitant.

 

DNA solution is the stable existence of DNA in a hydrated state.

When ethanol is added, the ethanol will deprive the water molecules around the DNA, causing the DNA to lose water and easily polymerize.

In general experiments, double the volume of absolute ethanol is mixed with DNA, and the final content of ethanol accounts for about 67%.

 

 

Do I need to add NaAc or NaCl to ethanol precipitation of DNA?

When precipitating DNA with ethanol, why must NaAc or NaCl be added to the final concentration of 0.1~0.25mol/L?

 

In a solution with a pH of about 8, DNA molecules are negatively charged. Add a certain concentration of NaAc or NaCl to neutralize the negative charges on the DNA molecules and reduce the repulsive force of the same-sex charges between the DNA molecules, which is easy to aggregate with each other. DNA sodium salt precipitation is formed.

When the concentration of the added salt solution is too low, only part of the DNA will form DNA sodium salt and polymerize, which will cause incomplete DNA precipitation. When the added salt solution concentration is too high, the effect is not good. . In the precipitated DNA, due to the presence of excessive salt impurities, which affect the DNA digestion and other reactions, it must be washed or re-precipitated.

 

 

 

 

Nucleic Acid Knowledge Series (1)

(source:internet, reference only)


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