S-adenosylmethionine, the methyl donor used by DNA methylases.
Double-stranded DNA containing the recognition site of a Type IIe restriction enzyme that activates cleavage of slow and resistant sites by binding to a distal, noncatalytic site on the enzyme.
Ambiguity of Recognition
When more than one nucleotide is possible at a particular position in the recognition site of a restriction enzyme (degeneracy).
Bipartite Recognition Sequence
An interrupted, nonpalindromic recognition sequence.
Blue/White Cloning Assay
A restriction enzyme quality control assay developed at Promega to ensure the lowest possible incidence of false positives in cloning experiments. This assay mimics a cloning experiment and is able to detect the loss of a single nucleotide from the end of a linearized plasmid.
Bovine Serum Albumin (BSA) is used as a carrier and stabilizing protein in restriction enzyme digests. Promega’s BSA has been acetylated to ensure that it contains no interfering activities.
The specific double-stranded DNA sequence recognized by a restriction enzyme (same as recognition sequence).
Two or more of the same linear DNA molecule covalently linked end-to-end.
A restriction enzyme quality control assay for exonuclease and phosphatase contamination. DNA is cut with a restriction enzyme, ligated with T4 DNA Ligase and recut with the enzyme to demonstrate the integrity of the restriction fragment ends.
Methylation at the N6 position of adenine in the sequence GATC by dam methylase, an enzyme found in most laboratory strains of E. coli. dam methylation may prevent cleavage by some restriction enzymes.
Methylation at the C5 position of the internal cytosine in the sequence CCAGG or CCTGG by dcmmethylase, an enzyme found in most laboratory strains of E. coli. dcm methylation may prevent cleavage by some restriction enzymes.
When more than one nucleotide is possible at a particular position in the recognition site of a restriction enzyme (ambiguity of recognition).
Dinucleotide (or Trinucleotide) Bias
An increased or decreased probability of encountering a particular dinucleotide (or trinucleotide) sequence in a genome, used to predict the expected cut frequency of a restriction enzyme.
An enzyme that cleaves single- or double-stranded DNA at interior positions. Endonuclease activity may be specific (e.g., restriction enzymes) or nonspecific (e.g., nickases).
An enzyme that removes individual nucleotides from the end of single- or double-stranded DNA, usually specific for a 3´ or 5´ end.
Fidelity of Recognition
The stringency with which a restriction enzyme discriminates between its recognition site and closely related sites. Fidelity of recognition is significantly reduced under suboptimal conditions, leading to star activity.
Promega enzymes whose performance in digesting bacterial genomes has been demonstrated.
DNA that has been methylated on only one strand of a restriction enzyme recognition site.
Endonucleases encoded by genes with mobile, self-splicing introns or inteins (protein introns). Also known as intron or intein encoded endonucleases.
A restriction enzyme recognition sequence containing a dyad axis of symmetry separated by a specific number of totally degenerate nucleotides (denoted by the letter N).
Endonucleases encoded by genes with mobile, self-splicing introns or inteins (protein introns). Also known as homing endonucleases.
Restriction enzymes that have the same recognition sequence but may or may not share the same cut site.
The process by which restriction enzymes diffuse along a DNA molecule to find their recognition site.
A subset of isoschizomers that share the same recognition sequence, but cleave the DNA at a different site within that sequence.
An enzyme that makes a single-stranded break in double-stranded DNA, usually in a nonspecific manner.
A restriction enzyme recognition sequence without a dyad axis of symmetry. Non-palindromic sequences are typical of Type IIs enzymes.
A restriction enzyme quality control assay for exonucleases, other endonucleases and star activity. In this assay, increasing amounts of enzyme are added to a series of tubes containing substrate DNA. After a 16-hour incubation under appropriate conditions, the maximum number of units giving a clear, sharp, normal banding pattern is determined by agarose gel electrophoresis.
A restriction enzyme recognition sequence containing a dyad axis of symmetry using only A, C, G and T, i.e., the sequence reads the same in the 5´→ 3´ direction on each strand.
A restriction enzyme recognition sequence containing a dyad axis of symmetry with degenerate positions. For example, the recognition site of Sty I is given as CCWWGG; therefore, Sty I cleavage occurs at the sequences CCAAGG, CCATGG, CCTAGG and CCTTGG.
An enzyme that removes a phosphate from a nucleic acid or protein. Phosphatases are potential contaminants of restriction enzyme reactions and can reduce subsequent ligation efficiency.
Modification (R/M) System
A system composed of a restriction endonuclease and a methylase that share the same recognition sequence. The methylase modifies the host DNA, protecting it from the action of the restriction endonuclease, which cleaves unmodified (foreign) DNA. The methylase and endonuclease genes are usually adjacent to each other on the host DNA. Occasionally a third open reading frame is also present, believed to code for a “C”, or control, protein for endonuclease expression.
Recognition Site Density
When determining the number of units of restriction enzyme needed for a digest, consideration must be given to the number of recognition sites in 1µg of the sample DNA compared with the number of recognition sites in 1µg of the unit definition DNA. This is especially important when digesting PCR fragments and oligonucleotides and is occasionally a factor affecting plasmid digests.
A restriction enzyme recognition sequence that is difficult to cleave using the appropriate reaction conditions using a small excess of enzyme. No additional cleavage is realized by increasing the amount of enzyme used.
A map of a DNA sequence showing the restriction sites of one or more restriction enzymes.
The phosphate at which the DNA backbone is cleaved by a restriction endonuclease. Restriction enzymes hydrolyze the diester bond to yield 5´-phosphate and 3´-hydroxide DNA fragments.
A restriction enzyme recognition sequence that is partially cleaved under appropriate reaction conditions using a small excess of enzyme. Additional cleavage is realized by increasing the amount of enzyme used.
Undesired restriction enzyme cleavage at sites resembling the recognition sequence. This activity is accelerated under suboptimal reaction conditions, e.g., improper ionic strength, wrong divalent metal cofactor or the presence of volume excluders.
Reaction mechanism of restriction enzymes where the 3´-phosphate adjacent to the scissile phosphate plays a critical, but as yet not completely understood, role in catalysis.
Promega Type IIe restriction enzymes provided with a Reaction Buffer containing a noncleavable affector sequence that facilitates efficient digestion of slow and resistant sites.
Restriction enzymes consisting of a multimeric complex that contains restriction, methylation and specificity subunits. Recognition sites are bipartite and interrupted. Cleavage is distant and variable from the recognition site. AdoMet, ATP (hydrolyzed) and Mg2+ are required for cleavage. Not commercially available.
Restriction enzymes consisting of a homodimer. Recognition sites are palindromic, partially palindromic or interrupted palindromes. Cleavage is defined and within the recognition sequence. Only Mg2+ is required as a cofactor. These are the most common commercially available restriction enzymes.
Restriction enzymes consisting of a heterotrimer. Recognition sites are bipartite and interrupted. Both strands on both sides of the recognition site are cleaved, excising the recognition site. AdoMet and Mg2+ are required. Bcg I is the only commercially available enzyme of this type.
Restriction enzymes that would be classified as Type II or Type IIs but demonstrate slow or resistant sites. Efficient cleavage at these sites can be achieved by the binding of another (affector) recognition sequence to a distal, noncatalytic site on the enzyme.
Restriction enzymes that primarily exist in monomeric form. Recognition sites are nonpalindromic, nearly always contiguous and without ambiguities. At least one strand is cleaved outside the recognition sequence. Only Mg2+ is required as a cofactor. Many are commercially available.
Restriction enzymes that consist of restriction- and methylation-specificity subunits. Recognition sites are non-palindromic. Cleavage is approximately 25 bases from the recognition site. AdoMet, ATP (non-hydrolyzed) and Mg2+ are required. Not commercially available.
One unit is the amount of restriction enzyme necessary to completely digest 1µg of a specific DNA substrate, usually lambda, in one hour, in a 50µl reaction volume and at a specified temperature. Digestion is determined by ethidium bromide-stained agarose gel electrophoresis.
For restriction enzymes, buffer components such as glycerol and polyethylene glycol that reduce the amount of water at the enzyme-DNA interface. Volume excluders often cause increased star activity.