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Epicentre Forum 3 (1)
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Epicentre offers a variety of ligases for the covalent
attachment of the 5´-phosphate of one nucleic acid to the 3´-hydroxyl
of another. Ampligase® Thermostable DNA Ligase is used for ligation
amplification (LCR),1-5 Repeat
Expansion Detection,6-8 high
fidelity gene synthesis,9,10 and
for the creation of either single or multiple specific mutations.11 T4
DNA Ligase is typically used for cloning DNA fragments. T4 RNA Ligase
can be used for 5´-end tagging to map or sequence the 5´-ends
of RNA, facilitating cDNA synthesis, and for 3´-end labeling of
RNA. This article provides answers to the most common questions we receive
on ligases. If you have other questions related to these products, please
contact Epicentre's Technical Services Department at 800-284-8474.
Q: What is the optimal reaction temperature of Ampligase
DNA Ligase?
A: The enzyme is active over a wide range of temperatures
from 28°C to 85°C. While the optimum temperature for activity
is 70°C, the optimal reaction temperature is limited by the Tm of
the oligonucleotides used in the application. Temperatures above 70°C
can be used if the DNA structure will remain stable at those temperatures.
Q: How is the Ampligase DNA Ligase reaction different
from the T4 DNA Ligase reaction that catalyzes covalent attachment
of blunt DNA or DNA with short overhangs?
A: At the low temperatures required to anneal short
overhangs, Ampligase DNA Ligase is much less active than T4 DNA Ligase.
Ampligase DNA Ligase will not ligate blunt ends.
Q: How much Ampligase DNA Ligase is required for
a reaction?
A: Five units per 25 µl reaction is a good
starting point for most applications, however, each reaction must be
optimized. Excess enzyme may increase background. Too little enzyme may
result in low yields of ligation products in certain applications. Refer
to the given applications references for further recommendations.
Q: Does Ampligase DNA Ligase have other activities
similar to T4 RNA Ligase which can be used to ligate RNA to RNA or
RNA to DNA?
A: At Epicentre, we have not detected any RNA ligation
activity with Ampligase DNA Ligase.
Q: Can Ampligase DNA Ligase be used in DNA polymerase
buffers?
A: Ampligase DNA Ligase has very flexible buffer
requirements, and will work in most DNA polymerase buffers. However,
NAD must be added as a cofactor and is included in the Ampligase 10X
Reaction Buffer.
Q: To detect a point mutation using ligation amplification,
how close must the point mutation be to the ligation site?
A: We recommend that the point mutation be positioned
at the ligation junction. The enzyme will detect a mutation one base
away from the junction. However, at two bases away from the ligation
junction, the discrimination between matches and mismatches drops dramatically.
At three or more bases away, the mutation has little effect on ligation.
Q: What is Repeat Expansion Detection (RED) and
how is Ampligase DNA Ligase used in this procedure?
A: RED is a ligation-based method of genetic screening
that detects DNA regions composed of multiple trinucleotide repeats.6-8 Ampligase
DNA Ligase is used to generate multimers of trinucleotide repeat oligonucleotide
probes when cycled with a genomic DNA template. The size of the repeat
region can be determined by the size of the Ampligase DNA Ligase reaction
product. In the human genome, such "repeat expansion" within coding or
regulatory sequences has been implicated in genetic diseases.
Q: What is the thermostability of Ampligase DNA
Ligase?
A: The half-life of Ampligase DNA Ligase is 48
hours at 65°C and greater than one hour at 95°C. The enzyme has
been shown to be active for at least 500 thermal cycles (94°C/80°C)
or 16 hours of cycling.6
Q: What are the best reaction conditions for T4
DNA Ligase when cloning DNA with cohesive overhangs or DNA with blunt
ends?
A: For DNA with cohesive overhangs, we recommend
1X T4 DNA Ligase Buffer (33 mM Tris-Acetate, pH 7.8, 66 mM potassium
acetate, 10 mM magnesium acetate, 0.5 mM DTT), 0.5-1.0 mM ATP and a molar
ratio of 2:1 insert to vector DNA in a total volume of 15 µl. For
blunt ligations, the insert to vector ratio should be 10:1 in the above
buffer conditions. In both cases, the incubation should be performed
at room temperature. For best results, a blunt ligation should incubate
overnight, while cohesive overhang ligations are usually complete in
one hour. Two units of ligase are sufficient for most ligations, but
for maximum efficiency in blunt ligations, Epicentre sells T4 DNA Ligase
at 10 U/µl and recommends 10 U per reaction.
Q: The DNA ligation appears successful by gel analysis,
but my transformation efficiencies are much lower than I expected.
What is happening?
A: For maximum transformation efficiencies it is
necessary to heat-kill the ligase for 15 minutes at 65°C. If this
is not done, the ligase can coat the DNA and inhibit transformation.12 This
is especially true when using 10 U/µl T4DNA Ligase.
Q: What is the efficiency of ligation when using
T4 RNA Ligase?
A: The efficiency of ligation depends on the reaction.
When ligation is being used to create circular RNA intramolecularly from
uncapped RNA, we have seen efficiencies approaching 100%. Intermolecular
ligations generally give 50% ligation under standard conditions.
Q: Can T4 RNA Ligase be used to make substrates
for cDNA synthesis?
A: Yes, the enzyme can be used for preparation
of cDNA synthesis substrates.13-18 The
addition of an oligonucleotide to the 5´-end of mRNA can facilitate
synthesis of full-length cDNA.
Q: What conditions will ensure maximum ligation
using T4 RNA Ligase?
A: The condition of the ends of the nucleic acids
is very important. For example, when a 5´ oligo is added to decapped
mRNA, the mRNA molecule (the "donor") must have a 5´-monophosphate
and the oligo-nucleotide (the "acceptor") must have a 3´-hydroxyl
group. Both groups must not be made inaccessible to the enzyme due to
secondary structure. The reaction should contain a 20- to 100-fold excess
of the oligonucleotide to favor intermolecular ligation of the mRNA to
the oligonucleotide over intramolecular ligation. Oligoribonucleotide
acceptor molecules are strongly preferred by the enzyme. Oligodeoxyribonucleotide
acceptors decrease reaction efficiencies 100-fold. However, oligodeoxyribonucleotide
acceptors can be used efficiently if the final three bases of the oligo
are ribonucleotides. For both RNA and DNA oligonucleotide acceptors,
the reaction efficiencies are highest if the last three 3 bases are adenosines.
The presence of uridine in the last three bases will be inhibitory.
- Landegren, U. et al. (1988) Science 242, 229.
- Wu, D.Y. and Wallace, R.B. (1989) Genomics 4, 560.
- Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88, 189.
- Birkenmeyer, L. and Armstrong, A.S. (1992) J. Clin. Micro. 30,
3089.
- Dille, B.J. et al. (1993) J. Clin. Micro. 31,
729.
- Schalling, M. et al. (1993) Nature Genetics 4,
135.
- Schalling, M. et al. (1994) Genome Mapping and Sequencing
Meeting Abstracts, Cold Spring Harbor Laboratory, New York, 236.
- Sirugo, G. and Kidd, K.K. (1995) Epicentre Forum 2(3),
1.
- Sutton, D.W. et al. (1992) Transgenic Research 1,
228.
- Sutton, D.W. et al. (1995) Epicentre Forum 2(2),
1.
- Rouwendall, G.J.A. et al. (1993) BioTechniques 15,
68.
- Michelsen, B.K. (1995) Anal. Biochem. 225, 172.
- Volloch, V. et al. (1994) Nuc. Acids Res. 22,
2507.
- Fromont-Racine, M. (1993) Nuc. Acids Res. 21, 1683.
- Maruyama, K. and Sugano, S. (1994) Gene 138, 171.
- Liu, X. and Gorovsky, M.A. (1993) Nuc. Acids Res. 21,
4954.
- Volloch, V. et al. (1991) Proc. Natl. Acad. Sci USA 88,
10671.
- Kato, et al. (1994) Gene 150, 243.
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