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Epicentre Forum 2 (1)
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Epicentre Technologies offers several DNase enzymes with
different specificities for use in a variety of molecular biology techniques.
The activities and some suggested applications for each of these nucleases
are discussed below. All of Epicentre's DNases are rigorously purified
and tested to ensure the absence of exogenous nuclease activities.
Eliminates cloning artifacts due to contamination of plasmids
or cosmids with bacterial chromosomal DNA.
Plasmid-Safe DNase is a selective DNase, often used for
the removal of contaminating bacterial chromosomal DNA from preparations
of plasmid or cosmid clones. It has a unique nuclease activity that digests
linear double-stranded DNA and both linear and closed circular single-stranded
DNA in both 5´-3´ and 3´-5´ directions. However, the
enzyme has no activity on the supercoiled or nicked-circular forms of
double-stranded plasmid or cosmid vectors (Figure 1).
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Figure 1. Use of Plasmid-Safe ATP-Dependent DNase to remove
contaminating linear DNA. A mixture containing Sma I-digested
bacterial chromosomal DNA and intact plasmid DNA was incubated
with 10 U of Plasmid-Safe for 30 min at 37°C. Aliquots of the
reaction were examined by electrophoresis on a 1% agarose gel and
the gel was stained with ethidium bromide. Lane 1, kilobase ladder
DNA markers; lane 2, 3 mg of Sma I-digested bacterial chromosomal
DNA; lane 3, 500 ng of uncut plasmid DNA, lane 4, mixture of 3
mg of Sma I-digested bacterial chromosomal DNA and 500 ng
of uncut plasmid before Plasmid-Safe DNase treatment; lane 5, Mixture
of chromosomal DNA and plasmid DNA after incubation with Plasmid-Safe
DNase as described. Plasmid-Safe DNase digests linear chromosomal
DNA while leaving supercoiled plasmid DNA intact. |
Plasmids or cosmids prepared by most standard methods,
even cesium chloride gradient banding, are often contaminated with residual
amounts of host bacterial chromosomal DNA. This may result in inadvertently
subcloning, characterizing or sequencing the contaminating DNA rather
than the desired target. Removing chromosomal DNA contamination is especially
critical when using low-copy-number vectors for which chromosomal DNA
makes up a larger portion of the total DNA preparation. However, Plasmid-Safe
DNase used as a final treatment step for any plasmid or cosmid isolation
can save time and avoid problems. Additionally, treatment with Plasmid-Safe
DNase enriches for circular forms of plasmids or cosmids, which yield
the highest transformation efficiencies.
Following its use, Plasmid-Safe DNase can be heat-inactivated
by incubation at 70°C for 15 min. For this reason, it is sometimes
preferred over DNase I for removal of a linear DNA template following
in vitro transcription reactions. ATP, Mg++, and a neutral
pH are required for full enzymatic activity. Epicentre supplies both
an optimized 10X Reaction Buffer and a 10 mM ATP Solution with Plasmid-Safe
DNase.
Digests residual primers and other single-stranded DNA
Exonuclease I (Exo I) is a single-strand-specific exonuclease
that digests DNA in a 3´-5´ direction (Figure 2). Enzyme activity
requires Mg++ and a free 3´-hydroxyl terminus. The enzyme
is active under a wide variety of buffer conditions, and can be added
directly to many reaction mixes.
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Figure 2. Substrate specificity of Exonuclease I. 200 ng
of pUC19 DNA linearized with EcoR I, and 1 mg of a 100 mer
single-stranded oligonucleotide were mixed in 1X TA Buffer (33 mM
Tris-acetate pH 7.8, 66 mM K-acetate, 10 mM Mg-acetate, and 0.5 mM
DTT) and incubated at 37°C for 20 min in the absence or presence
of 10 U of Exo I. Reaction products were separated by electrophoresis
on a 1% agarose gel. Lane 1, molecular weight markers; lane 2, minus
Exo I treatment; lane 3, plus Exo I treatment. Exonuclease I completely
digested the linear single-stranded oligonucleotide while leaving
the linear double-stranded plasmid DNA intact. |
Exo I conveniently removes residual primers and single-stranded
DNA from amplification reactions, creates blunt ends from 3´-overhangs,
and removes single-stranded DNA from nucleic acid mixtures.
Used for generation of nested deletions, for site-directed
mutagenesis, or other applications. Available alone or in the Discrete-Delete
Deletion Kit (This kit has been discontinued*).
Exonuclease III (Exo III) will digest one strand of a double-stranded
DNA molecule, starting from a blunt end, 5´-overhang, or nick. The
enzyme acts in a 3´-5´ direction, and proceeds at a uniform rate
along the DNA template. Timed aliquots from an Exo III digestion contain
stretches of single-stranded DNA of predictable and reproducible lengths.
If desired, these single-stranded regions can be removed using a single-strand-specific
nuclease, such as Mung Bean Nuclease (see below), to obtain a double-stranded
deletion (Figure 3).
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Figure 3. Generation of nested deletions using the Discrete-Delete
Kit . 5 mg of pUC19 DNA, digested to completion with Kpn I
and Hind III restriction endonucleases, was incubated at
30°C with 500 U of Exonuclease III in 100 ml 1X TA buffer (33
mM Tris-acetate pH 7.8, 66 mM K-acetate, 10 mM Mg-acetate, and
0.5 mM DTT). 10 ml aliquots were removed at 30 sec intervals and
digested with 15 U Mung Bean Nuclease at 37°C for 30 min according
to the kit protocol. Reaction products were separated by agarose
gel electrophoresis and stained with ethidium bromide. MW, molecular
weight markers; Lane 1, 0 sec; lane 2, 30 sec; lane 3, 60 sec;
lane 4, 90 sec; lane 5, 120 sec; lane 6, 150 sec. |
The primary use for Exo III is in conjunction with Mung
Bean Nuclease or another single-strand-specific nuclease for generation
of unidirectional sets of nested deletions for structure-function studies
or dideoxy sequencing.1 Exo III
can also be used for site-directed mutagenesis protocols,2 and
for generating single-stranded DNA templates for sequencing.3
The Discrete-Delete Deletion Kit*,
a cost-effective kit for generating nested deletions, contains Exonuclease
III, Mung Bean Nuclease, T4 DNA Ligase, reaction buffers, ATP Solution
and a detailed protocol for the easy and convenient generation of deletion
clones from up to 50 mg of DNA.
Digests single-stranded DNA or RNA with greater specificity
than S1 Nuclease.
Mung Bean Nuclease is a single-strand-specific nuclease
which degrades single-stranded DNA or RNA (Figure 4). The enzyme does
not, however, digest double-stranded DNA, double-stranded RNA, or DNA/RNA
hybrids. Mung Bean Nuclease is preferable to S1 Nuclease for most applications
because of its lower intrinsic activity on duplex DNA.
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Figure 4. Specificity of Mung Bean Nuclease for single-stranded
DNA. 3 mg of Hind III-linearized pBSC DNA was treated
with 300 U Exonuclease III according to the Discrete-Delete™ Kit
procedure. At 3 successive 1 min intervals following addition of
Exo III, duplicate aliquots were removed. One aliquot from each
set was treated with 10 U Mung Bean Nuclease according to the Discrete-Delete
Kit procedure, while the second aliquot was not treated (control).
Samples were then electrophoresed on a 1% agarose gel and stained
with ethidium bromide. Lane 1, kb markers; lanes 2-4, control samples
(1 min, 2 min, 3 min, respectively); lanes 5-7, treated samples
(1 min, 2 min, 3 min, respectively). The control samples contain
both single-stranded (ss) and double-stranded (ds) DNA. The treated
samples show that Mung Bean Nuclease digested only the ss-DNA,
leaving the ds-DNA intact. |
Mung Bean Nuclease may be used in a variety of applications.
These include high resolution mapping of termini and exon structure of
RNA transcripts,4 restriction site
modification or removal by digestion of protruding single-stranded ends,1 and
unidirectional deletion of DNA sequences (in combination with ExoIII)3 to
facilitate subcloning of discretely-sized deletion mutants.
Mung Bean Nuclease is available alone, or as a component
in the Discrete-Delete Exo III/Mung Bean Nuclease Deletion Kit. *
May be used for complete removal of all DNA in a reaction
mixture, for DNA Footprinting, or for Nick Translation.
Epicentre's RNase-free DNase I is an endonuclease active
on all free forms of single and double-stranded DNA, hydrolyzing it to
a mixture of short oligo- and mononucleotides. The enzyme requires Mg++ for
activity, and is active under a wide variety of buffer conditions.
DNase I is used when the removal of all DNA in a reaction
mixture is desired. For example, it is frequently used to remove DNA
template molecules from an in vitro transcription reaction (Figure 5).
Diluted DNase I is also frequently used in conjunction with a DNA polymerase
to label DNA by nick translation.5
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Figure 5. DNA removal from in vitro transcription reactions
using RNase-free DNase I. The linearized DNA Control from an
Epicentre T7 Transcription Pac-Kit™ was transcribed using
T7 RNA Polymerase according to standard conditions. Lane 1, kb
ladder; lane 2, DNA control; lane 3, transcription mixture; lane
4, transcription mixture treated with 1 U DNase I for 15 min at
37°C. |
DNase I is not active on DNA bound to proteins. This property
is often exploited in "footprinting experiments" to study protein-DNA
interactions.6
- Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd
edition), Cold Spring Harbor Laboratory Press, New York.
- Vandeyar (1988) Gene 65: 129.
- Henikoff (1984) Gene 28: 351 .
- Murray et al. (1986) Anal. Biochem. 158: 165.
- Rigby et al. (1977) J. Mol. Biol. 113: 237.
- Gales and Schmitz (1978) Nuc. Acids Res. 5: 3157.
*The Discrete-Delete Kit is no longer available.
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