| AdoMet |
S-adenosylmethionine, the methyl donor used by DNA
methylases. |
| Affector Sequence |
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. |
| BSA (acetylated) |
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. |
| Cognate Sequence/Site |
The specific double-stranded DNA sequence recognized by a
restriction enzyme (same as recognition sequence). |
| Concatamers |
Two or more of the same linear DNA molecule covalently linked
end-to-end. |
| Cut-Ligate-Recut Assay |
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. |
| dam Methylation |
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. |
| dcm Methylation |
Methylation at the C5 position of the internal
cytosine in the sequence CCAGG or CCTGG by dcm methylase, an enzyme found in most
laboratory strains of E. coli. dcm methylation may prevent cleavage by some
restriction enzymes. |
| Degeneracy |
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. |
| Endonuclease |
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). |
| Exonuclease |
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. |
| Genome Qualified |
Promega enzymes whose performance in digesting bacterial
genomes has been demonstrated. |
| Hemi-methylated
DNA |
DNA that has been methylated on only one strand of a
restriction enzyme recognition site. |
| Homing
Endonucleases |
Endonucleases encoded by genes with
mobile, self-splicing introns or inteins (protein introns). Also known as intron or intein
encoded endonucleases. |
| Interrupted Palindrome |
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). |
| Intron or Intein
Encoded Endonucleases |
Endonucleases encoded by genes with
mobile, self-splicing introns or inteins (protein introns). Also known as homing
endonucleases. |
| Isoschizomers |
Restriction enzymes that have the same recognition sequence
but may or may not share the same cut site. |
| Linear Diffusion |
The process by which restriction enzymes diffuse along a DNA
molecule to find their recognition site. |
| Neoschizomers |
A subset of isoschizomers that share the
same recognition sequence, but cleave the DNA at a different site within that sequence. |
| Nickase |
An enzyme that makes a single-stranded break in
double-stranded DNA, usually in a nonspecific manner. |
| Non-palindromic |
A restriction enzyme recognition sequence without a dyad axis
of symmetry. Non-palindromic sequences are typical of Type IIs enzymes. |
| Overdigest Assay |
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. |
| Palindromic |
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´à 3´ direction on each strand. |
| Partially Palindromic |
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. |
| Phosphatase |
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. |
Restriction/
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. |
| Resistant Sites |
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. |
| Restriction Map |
A map of a DNA sequence showing the restriction sites of one
or more restriction enzymes. |
| Scissile
Phosphate |
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. |
| Slow Sites |
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. |
| Star Activity |
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. |
| Substrate-Assisted Catalysis |
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. |
| Turbo™
| Promega Type IIe restriction enzymes
provided with a Reaction Buffer containing a noncleavable affector sequence that
facilitates efficient digestion of slow and resistant sites. |
| Type I
Restriction Enzymes |
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. |
| Type II
Restriction Enzymes |
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. |
| Type IIb
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. |
| Type IIe
Restriction Enzymes |
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. |
| Type IIs
Restriction Enzymes |
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. |
| Type III
Restriction Enzymes |
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. |
| Unit Definition |
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. |
| Volume Excluders |
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. |
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