What Is an Endonuclease?

Endonuclease is an enzyme that can hydrolyze the phosphodiester bond inside the molecular chain to generate oligonucleotides. It corresponds to exonuclease. From the specificity of the substrate, it can be divided into DNase, DNase and other enzymes that break down DNA; RNase, RNaseT1 and other enzymes that break down RNA. In general, most are not base-specific, but there are also enzymes such as spleen RNase, RNaseT1, or restriction enzymes that can recognize and cut specific bases or base sequences.

Endonuclease

Endonuclease is an enzyme that can hydrolyze the phosphodiester bond inside the molecular chain to generate oligonucleotides. It corresponds to exonuclease. From the specificity of the substrate, it can be divided into DNase, DNase and other enzymes that break down DNA; RNase, RNaseT1 and other enzymes that break down RNA. In general, most are not base-specific, but there are also enzymes such as spleen RNase, RNaseT1, or restriction enzymes that can recognize and cut specific bases or base sequences.

Chinese name

Endonuclease

Foreign name

endonuclease

Specific classification

DNase, DNase

Nature

Mostly not base specific

More than 30 years ago, when people were

The same DNA is cleaved with different restriction enzymes to obtain the cleavage sites of various restriction enzymes. The site maps thus established are helpful for analyzing the structure of DNA.

Restriction endonuclease analysis technology is an effective method for pathogen mutation, virus identification, typing, understanding of genetic structure, and epidemiological research. It has important practical significance for animal quarantine, especially for distinguishing entry and exit animals and animals. Whether the product carries a virus is a vaccine or a wild virus, and it is of great significance to infer whether it is a local or foreign virus.

Restriction endonucleases (hereinafter referred to as restriction enzymes) are a class of DNA hydrolases that recognize specific nucleotide sequences in double-stranded DNA. They hydrolyze DNA in an endogenous manner to produce 5'-P and 3'-OH ends. In 1952, Luria et al. And Bertani et al. (1953) discovered host-controlling phenomena when studying phages. Arber and colleagues demonstrated with radioisotope labeling that damage to the phage in the new strain was accompanied by degradation of its DNA, but that the host's own DNA did not degrade, and based on this they proposed the restriction-modifying enzyme hypothesis. For a host cell, restriction enzymes and DNA methylases are a pair of enzymes in their cells. They have the same recognition sequence for DNA substrates, but have opposite biological functions. The function of restriction enzymes is in the DNA molecule. Internal disassembly and hydrolysis, methylase is modified, DNA molecules can be modified to avoid the recognition of restriction enzymes, and methylases only modify the host's own DNA, thereby avoiding the damage of the restriction enzyme to its own DNA.

Restriction enzymes are mainly divided into three types: Type restriction enzymes are complex function enzymes with two functions of restriction and modification, but there is no fixed cleavage site on the DNA strand. Generally, the restriction enzyme is 1 kb to a few kb from the cleavage site Places are randomly cut and do not produce specific fragments. Type enzymes are basically similar to type enzymes, except that type enzymes have specific cleavage sites, but these two types of enzymes have little significance for DNA digestion analysis. Commonly referred to as restriction enzymes are Type II enzyme, which can recognize and cleave specific nucleotide sequences on the DNA strand, to produce specific DNA fragments.

2. Recognition sequence and recognition structure of the end product of the digestion product. Most of the recognition sequences have biaxially symmetric structures or palindromes. For example, the recognition sequence of EcoRI is:

GAA

TTC

Horizontal axis

CTT

AAG

Vertical axis

If the sequence on one side of the vertical axis is rotated by 180 ° with the horizontal axis as the center, the sequences on both sides of the vertical axis are symmetrical to each other. This structure is also called a double symmetrical structure. The recognition sequence for most enzymes is 4-6 nucleotides in length. The 4 nucleotide sequence appears in the DNA strand with high frequency. For a randomly arranged DNA molecule, the theoretical value is 1/44. Therefore, the restriction enzyme of the 4 nucleotide recognition sequence cuts many points on the DNA strand, resulting in The number of fragments is large and the length is short, showing that the specificity of the enzyme is low. For enzymes with 5 and 6 nucleotide recognition sequences, the frequency of occurrence is 1/45 and 1/46, respectively. Therefore, the frequency of 6 nucleotide sequences in DNA is low, the specificity of the enzyme is strong, and 8 nucleotides are recognized. Sites have a lower probability of appearing in the DNA strand (1/48), are more specific, and can provide longer DNA fragments. Some restriction enzymes have atypical biaxially symmetric sequences. The palindrome recognition sequence is separated by one or several other nucleotides, such as Bgl. This enzyme is more specific than the typical palindrome sequence of the same length. The enzyme is slightly higher. In addition, there are some restriction enzymes (about 10 types, such as BbV, etc.) whose recognition sequences do not show palindrome structure. When they degrade double-stranded DNA, most of the restriction points are not within the recognition sequence, but are 5 to 5 away from the recognition sequence. 13 nucleotide residues vary.

The end structure of restriction fragments: not only does the restriction enzyme have a specific recognition sequence, but any kind of enzyme will always hydrolyze the 3 'and 5'-phosphate diester bond 3' phosphate when cutting the DNA strand The ester bond makes the 5 'end of the product carry a phosphate monoester group, while the 3' end is a free hydroxyl group. Therefore, the ends of all products of an enzyme have the same structure. According to the structural characteristics of the tangency sequence, the ends of the products can be divided into two types: sticky ends and flat ends. Sticky ends refer to single-stranded structures with 1-4 nucleotide residues at the ends of DNA fragments after digestion. The protruding single-stranded ends at the ends of the fragments are complementary. There are two types of 5'- and 3'-sticky ends. The protruding single-stranded 5 'phosphate monoester is called the 5'-sticky end, while the protruding single-chain containing 3'-hydroxyl is called the 3'-sticky end. The blunt end refers to the structure of a blunt end after digestion. When DNA is reconstituted in vitro, sticky ends are effective substrates for DNA ligase and have high ligation efficiency.

The speed of restriction enzyme digestion has a great relationship with the nature of the substrate. The single- and double-stranded structure of the substrate, the configuration of the molecule, the number of enzyme recognition sites in the DNA strand, and the sequence near the site all affect the enzyme. Catalytic reaction. Covalently closed loop (supercoil configuration) DNA is slower than its corresponding linear molecule in enzymolysis. To completely degrade supercoil configuration DNA, a large amount of enzyme is required. For DNA-RNA hybrid double-strand digestion, when Molloy et al. Digested with 8 kinds of EcoRI, the DNA strand could be cut, but the amount of enzyme was 20-50 times larger than double-stranded DNA. Some restriction enzymes can also cleave single-stranded DNA, such as Hae III.

Each restriction enzyme has its own recognition specificity. Under normal enzyme reaction conditions, the specificity will not change, but under special conditions, the specificity of some enzymes will change accordingly, such as when the reaction buffer When the pH is increased from 7.5 to 8.5, or when the glycerol concentration exceeds 5%, or when Mn is used instead of Mg , the recognition sequence of EcoRI is changed from the original GAATTC to AATT. Therefore, under inappropriate reaction conditions, the restriction enzyme may exhibit a secondary activity that is different from the original specificity. Therefore, in the enzyme digestion analysis, it is necessary to ensure the enzymatic hydrolysis conditions, pay attention to the purity of the substrate DNA, and prevent the secondary activity of the enzyme as much as possible.

The concentration of the salt in the reaction solution is critical, so determine the concentration of the salt based on the enzyme used. Restriction enzymes do not have strict requirements for magnesium ions, and 5-30mmol / L can work. The amount of restriction enzyme is generally 1-5 units of DNA per microgram. It is advisable to use an excess of 3-5 times for insurance purposes, but it is best to perform a preliminary experiment first.

In theory, prolonging the reaction time can save the enzyme, but long-term digestion is restricted by factors such as the stability of the enzyme and the influence of miscellaneous enzymes. Domestic enzymes should not exceed 1.5 hours, and other enzymes are generally suitable for 1-2 hours. The temperature of the enzyme digestion reaction is generally 37 ° C. Only a few enzymes require special reaction temperatures, such as Taq, which requires 65 ° C. Most enzymes are still active below 37 ° C. For example, EcoRI is still active at 5 ° C. Many enzymes can be terminated by heat treatment (65 ° C) for 10-15 minutes, but not all enzymes can be thermally terminated. Enzymes that cannot be thermally terminated can be terminated with excess EDTA or with phenol extraction.

1. Operational steps Take animal virus as an example to illustrate the enzymatic digestion reaction step.

(1) Extraction and purification of viral DNA When the cells that reproduce the virus have 80% -100% cytopathic disease (CPE), harvest and freeze-thaw three times to rupture the cells to release the virus, centrifuge at 3 000 r / min for 10 minutes, and aspirate Clear, add 10% SDS to a final concentration of 1%, and act in a water bath at 56 ° C for 30 minutes, add an equal volume of saturated phenol, mix and centrifuge at 10 000 r / min for 10-15 minutes, and repeatedly extract the supernatant until there is no protein at the interface Take the supernatant, remove the phenol several times with ether, remove the ether under vacuum, and then add 2.5 times pre-cooled anhydrous ethanol to precipitate the nucleic acid. After centrifugation at 12 000 r / min, the nucleic acid precipitate is washed twice with 70% ethanol, pH 8.0 The TE buffer solution is dissolved. Take a small amount to measure its content and purity with a UV spectrophotometer, and store the rest at -20 ° C for later use.

(2) Enzymatic digestion The viral DNA was dissolved with TE to approximately 0.5 g / l. Then add 5l of 10 × enzyme digestion buffer (50mmol / L NaCl, 10mmol / L Tris-HCl pH7.8, 10mmol / L MgCl2, 1mmol / L DTT), 5l DNA, 2l restriction enzyme (1-5l / g DNA), make up to 50 l with sterilized double distilled water, gently shake on a shaker for a few seconds, and then slightly centrifuge (3,000 r / mmin) for a few seconds, so that the tube wall liquid is concentrated on the bottom of the tube, and incubated in a 37 ° C thermostatic water bath 1 hour, 65 ° C water bath for 10 minutes, or stop the reaction with EDTA.

(3) Electrophoresis Select different concentrations of agarose or polyacrylamide gels as supports for electrophoresis according to the size of the digested fragments. At the same time, add standard DNA as a molecular weight reference and stain with ethidium bromide (EB) or silver nitrate. Observe the results under the corresponding light source and record the electrophoresis spectrum.

The above is the basic process of the enzyme digestion reaction operation. If performing multiple digestion reactions, consider that the reaction buffer is basically suitable for the various enzymes used. If two or more enzymes have different requirements for the buffer solution, such as different salt concentration requirements, first perform a low-salt enzyme digestion reaction, and then perform a high-salt reaction. If the reaction temperature is not the same, first perform the low-temperature enzyme. Cut, and then perform high temperature digestion.

2. Restriction endonucleases play an extremely important role in molecular biology research. Restriction endonucleases can not be separated from almost every research field, and their applications are very extensive, such as DNA analysis of pathogenic microorganisms; DNA sequence analysis, cutting large DNA molecules into small fragments for sequence analysis; DNA recombination and construction of new plasmids; establishment of physical maps of DNA, etc.

Restriction EndonucleaseAnalysis REA has become a commonly used method. Digestion of DNA by enzyme digestion, and electrophoresis staining to show fragments of different sizes, analyze the mobility and number of these fragments, You can understand the certain characteristics of the genetic material of the pathogenic microorganisms. Based on this, using double-enzyme cutting or hybridization methods, you can infer the arrangement order of the fragments and the enzyme cutting sites, and infer the similarity or difference between the DNA. It is of great significance for the detection of animal viruses, especially for vaccine, wild and mutant strains.