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Restriction endonuclease digestion

Restriction endonuclease digestion

Standard reaction conditions for restriction endonucleases

One unit of a restriction endonuclease is defined as the amount of enzyme required to cleave 1 μg of substrate DNA in a 50-μl reaction mixture in 1 hr. The reactions should be carried out in the buffer and temperature optimized for each enzyme. The use of excess restriction endonucleases reduces the reaction time, or the amount of restriction endonucleases can be reduced by incubating the reaction for longer period of time (e.g., ~16 hr). In this case, it should be considered that star activity of a restriction endonuclease may become prominent due to the use of an excess amount or that the enzyme is not inactivated (see page 96) during the prolonged incubation.

 

Restriction endonucleases

Restriction endonucleases are recommended to be kept on ice or in a cold rack when removed from the freezer. They are usually added last to the reaction mixtures, and care should be taken so that the enzyme is evenly mixed with the other components. The amount of enzymes should be adjusted based upon the types of DNA to be cleaved.

 

DNA

Complete cleavage relies on the purity and concentration of substrate DNA. High concentrations of DNA the result in increased viscosity of the reaction mixtures, which interferes with free diffusion of the enzyme and DNA, reducing effective action of the restriction enzyme. Optimal concentration of DNA is ~0.02 to 0.2 μg/μl. Contaminants such as protein, phenol, chloroform, ethanol, EDTA, SDS, and CsCl can lead to reduced enzymatic activity. DNA nucleases degrade substrate DNA. DNA binding proteins present in substrate DNAs could interfere with binding of restriction endonucleases to DNA, thus inhibiting DNA cleavage by restriction endonucleases. Generally, purified DNA is dissolved in TE buffer (10 mM Tris-HCl/pH7.5, 1 mM EDTA). EDTA chelates divalent cations such as Mg2+ that is required for most nucleases including restriction endonucleases. For the storage of DNA, EDTA functions as a protective agent of DNA from contaminating nucleases. However, for cleavage of DNA with restriction endonucleases, care should be taken so that the concentration of EDTA in the digection mixture should be maintained for below that of Mg2+ required for optimal activity of a given restriction endonuclease. It is also important to consider that DNA methylation affects the cleavage of DNA by many restriction endonucleases.

Bacteriophage DNA, plasmid DNA, genomic DNA, and PCR products are common substrates of restriction endonucleases.

 

Lambda (λ) DNA

Lambda (λ) DNA is a relatively large (~48.5 kb) molecule of linear DNA, and is mostly used to measure activities of a number of different restriction endonucleases due to the presence of many different restriction sites. One unit of restriction endonucleases is the amount of enzyme required to cleave 1 μg of λDNA in a 50 μl of reaction mixture in 1 hr. The 12-nucleotide single-strand overhangs present at the termini of l DNA are complementary and can anneal to form a cos site. This often makes the digestion reaction look incomplete. Therefore, it is recommended to heat the reaction mixture at 65 for 5 min before gel electrophoresis when λDNA is digested with restriction endonucleases.

 

Plasmid DNA

Plasmid DNA, when purified, takes a covalently-closed-circular form with negative supercoiling and ranges from 3 to 15 kb in size. In contrast to linear DNA, high concentrations of restriction endonucleases are required for complete digestion of supercoiled plasmid DNA. The amount of restriction endonucleases required can be reduced, once supercoiled plasmid DNA is converted to a nicked or linear form of DNA by other means. In addition, the number of restriction sites within substrate DNA influences the amount of restriction endonucleases required for complete digestion.

 

Genome DNA

Methylated genome DNA with high viscosity is difficult to cleave by restriction endonucleases. If digestion is inhibited by methylation, try isoschizomers which recognize the same sequence but are not affected by methylation of DNA. Viscosity can be reduced by increasing the reaction volume. Diluted concentrations (0.05-0.2 μg/μl) of genomic DNA can be effectively cleaved by most restriction endonucleases. If dilution is difficult to achieve, heat DNA at 65 for 10 min and then add restriction endonucleases to reaction mixtures. This sometimes helps cleavage of genomic DNA by restriction endonucleases. It was reported that addition of 1-5 mM spermidine or 0.1 μg/μl BSA could increase cleavage efficiency by restriction endonucleases.

 

PCR products

PCR-amplified DNA fragments including the primer regions can contain recognition sequence of restriction endonucleases. The total number of restriction endonuclease recognition sequence in PCR products becomes greater with higher efficiency of PCR. Therefore, the amounts of restriction endonucleases and incubation period should be adjusted accordingly in proportion to the amounts of amplified DNA. Note that 1-3 or more bases are required beyond the ends of the restriction site of PCR products for efficient cleavage by restriction endonucleases. Cleavage of DNA is difficult or not possible if the restriction endonuclease recognition sequence is too close to the ends of the DNA. In addition, the proofreading activity of some PCR polymerases could degrade some nucleotides from 3' ends, impairing the integrity of the restriction endonuclease recognition sequence present near the ends of amplified DNA fragments. Using high concentration of dNTP and lowering the reaction temperature to 4 after PCR can help overcome this problem. An enormous number of restriction endonuclease recognition sites arises particularly if primer-dimers are amplified. The overwhelmingly large amounts of primer dimers result in greatly reduced cleavage of PCR products by restriction endonucleases. Purification of PCR-amplified DNA is recommended prior to restriction endonuclease digestion.

 

Reaction buffer

For each restriction endonuclease, 10X concentrated buffer is provided, and thus should be diluted 10-fold into the reaction mixture with some deionized water if needed, prior to DNA digestion. Selecting a buffer for a single enzyme is easy, but for double digestion, an appropriate buffer should be selected that can support activities of both enzymes. If there is hard to find such a buffer, for a particular double digestion, digestion of DNA should be carried out in a sequential manner one by one with the optimal buffer provided with each restriction endonuclease.

 

pH

Most enzymes have optimal pH between 7.2 and 8.5. Star activity can be observed with some restriction endonucleases if they are used outside the optimal pH range.

 

Mg2+

Mg2+ is a divalent metal ion, an essential cofactor required for restriction endonucleases activity. Cleavage activities of most restriction endonucleases are not greatly affected by varying concentrations of Mg2+; within a range; virtually no difference in enzyme activity is observed within a range from 5 to 30 mM.

 

Salt concentrations

Activity of restriction endonucleases is greatly affected by types of salt and their concentrations. Typically, activities of many restriction endonucleases are promoted by the presence of 50-150 mM NaCl or KCl, but some enzymes are inhibited with 20 mM or higher salt concentration. Some restriction endonucleases prefer acetate to chloride for their activity. Low salt concentration or unfavorable salts in reaction mixtures may result in star activity.

 

Bovine serum albumin (BSA)

Addition of BSA to storage solution or reaction mixtures increases stability of restriction endonucleases. BSA protects many enzymes including restriction endonucleases from proteolytic degradation by proteases, from nonspecific adsorption to reaction tubes, or from damage by heat or other contaminants. In general, 0.1 μg/μl BSA can increase enzyme activity by 1.5~6 folds. Addition of BSA does not interfere with restriction endonucleases that dose not require BSA for higher activity or stability.

 

Glycerol

Frequent cycles of freezing and thawing can reduce enzyme activity. The addition of glycerol to storage solution for restriction endonucleases prevents storage solution from freezing even at -20 The presence of 10% glycerol in reaction mixtures does not affect activity of most restriction endonucleases. For some restriction endonucleases, however, as low as 5% glycerol can reduce cleavage activities, and sometimes causes star activity.

 

Reaction mixtures

The relative amounts of restriction endonucleases, substrate DNA, and reaction volume should be decided based upon many factors such as purity of DNA, the number of restriction sites in the substrate, and viscosity of the reaction mixtures. Furthermore, reaction volumes should be adjusted so that final concentrations of salt and glycerol are within the range that does not cause significant inhibition or star activity. Use of too small reaction volumes should be avoided, since small-volume reactions are more susceptible to pipetting errors. Therefore, reaction volumes of 10-50 μl are recommended to digest 1 μg of DNA. To keep glycerol concentration less than 5% in reaction mixtures, the amount of restriction endonucleases, which are supplied in 50% glycerol, should not exceed 10% of the total reaction volume.

 

Mixing

Mixing is an important step for successful digestion, yet it is often overlooked. The reaction mixture must be thoroughly mixed to achieve complete digestion. Brief vortexing is recommended for complete mixing. Gentle pipetting of the reaction mixture or "flicking" of the reaction tube can be the alternative means of mixing, but make sure to completely mix the reaction contents. If necessary, spin down the mixture in a centrifuge for a short time prior to incubation.

 

Incubation temperature

The recommended incubation temperature for most restriction endonucleases is 37. Restriction endonucleases isolated from thermophilic bacteria require higher incubation temperatures ranging from 50 to 65. Refer to 1.3.5 Activity of thermophilic restriction endonucleases at 37.

 

Incubation time

Based on the unit definition above, 1-hr incubation is appropriate for most purposes. Incubation time can be shortened with excess amounts of restriction endonucleases, and the amounts of enzyme can be curtailed by increasing incubation time. It should be noted that longer incubation or excess amounts of enzyme can lead to undesirable consequences. Therefore, it is necessary to confirm how long the activity of a restriction endonuclease can last or how much enzyme can be used without displaying star activity.

 

Reaction termination

If no further digestion is necessary, the reaction can be terminated by adding the 6X stop solution (50% glycerol, 50 mM EDTA, 0.05% bromophenol blue) to 1X concentration. If further manipulation of the digested DNA is required, heat inactivation (mostly at 65-80 for 20 min, see page 94- 95) is the simplest method to stop the reaction. Heat treatment is not an effective means to inactivate some restriction endonucleases, particularly those from thermophilic bacteria. Extraction of digested DNA with phenol/chloroform followed by ethanol precipitation should be carried out in order to inactivate heat-resistant restriction endonucleases.

 

Storage

Storage at -20 is recommended for most restriction endonucleases. However for some enzymes, storage at -70 is recommended for periods longer than 30 days. BSA and 10X reaction buffers provided with the restriction endonucleases should also be stored at -20. Direct mixing of BSA with 10X reaction buffers should be avoided, or precipitation will occur.

 

Stability

Most restriction endonucleases are stable over 12 months at -20. Each restriction endonucleases produced by Enzynomics is inspected under rigorous quality control with regard to its activity and stability. Normally original activity can last even 1 year after sales. For further satisfaction of the customers, however, Enzynomics has an exchange policy; the unused amounts of old restriction endonucleases can be exchanged within one year after purchase with the equivalent amounts of a fresh enzyme without any additional cost.

 

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