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What are the advantages of using an RNase H minus form of reverse transcriptase?
M-MLV Reverse Transcriptase, RNase H Minus, is the enzyme of choice for construction of
full-length cDNA libraries.
Promega now offers two forms of M-MLV RT, RNase H Minus: A deletion mutant (Cat.# M5301)
and a point mutant (Cat.# M3682).
A reverse transcriptase (RT) is a type of DNA polymerase that catalyzes the synthesis
of a complementary DNA (cDNA) from an RNA template (1-3). The ability of reverse
transcriptases to make a cDNA copy of RNA is used in many different molecular biology
applications, including the production of cDNA libraries, RT-PCR(a),
primer extension and RNA sequencing.
The two most commonly used reverse transcriptases are the retroviral enzymes AMV RT,
purified from Avian Myeloblastosis Virus (4,5) and M-MLV RT purified from an E.coli
strain expressing a recombinant clone of Moloney Murine Leukemia Virus Reverse
Transcriptase (6,7). Both AMV and M-MLV RT possess two major activities: an RNA-dependent
DNA polymerase activity and an RNase H activity. The DNA polymerase activity of the
enzymes is essential for all applications requiring cDNA synthesis. However, the RNase H
activity of the enzymes is undesirable and interferes with the enzymes' ability to produce
full-length cDNA.
RNase H activity is an important consideration when choosing the most suitable reverse
transcriptase for cDNA synthesis. The RNase H activity of reverse transcriptases causes
the degradation of the RNA strand of an RNA:DNA hybrid much like the activity of E.
coli RNase H. When cDNA is synthesized, the RNA:DNA hybrids created can act as
substrates for the RNase H activity of reverse transcriptases. The effect of the RNase H
activity on cDNA synthesis is two-fold; both the total yield of cDNA and the percentage of
full-length cDNAs are effected. Reverse transcription is initiated from a DNA primer
hybridized to a specific sequence in the RNA template (often poly(A)). The primer:RNA
hybrid acts not only as a priming site for polymerization but also as a substrate for the
RNase H activity of the reverse transcriptase. The yield of cDNA synthesized is inversely
proportional to how much of the RNA:DNA hybrid is destroyed by the RNase H activity. In
some applications this can increase the minimum amount of RNA required to detect cDNA
synthesis. In addition, the RNase H activity of the enzyme can cut the RNA strand near the
site of DNA polymerization. If this occurs, the uncopied portion of the RNA molecule can
dissociate from the transcription complex, and cDNA synthesis will be terminated. The
longer the RNA molecule is, the more likely this will ocur. Long RNA molecules (>5kb)
are less likely to be fully copied into cDNA when the RNase H activity of the reverse
transcriptase is active.
The DNA polymerase activity of reverse transcriptases is not dependent on the
concomitant RNase H activity of the enzyme. As such, the deleterious effects of the RNase
H activity on cDNA synthesis can be eliminated by using a reverse transcriptase in which
the RNase H activity has been inactivated. This is done by introducing a mutation in the
RNase H domain of the protein, and such reverse transriptases are referred to as RNase H
minus. Elimination of the RNase acivity results in more efficient initiation of reverse
transcription as the primer:RNA hybrids are not degraded. In addition, elimination of
RNase H activity results in more efficient synthesis of full-length cDNAs since synthesis
is not prone to termination by RNA template degradation near the polymerization site. For
applications such as cDNA library synthesis where a high percentage of full-length cDNA is
desired, use of an RNase H minus reverse transcriptase is recommended.
Two types of RNase H minus reverse transcriptases are available from Promega. Both
enzymes are engineered forms of M-MLV reverse transcriptase. M-MLV Reverse Transcriptase,
RNase H Minus, Point Mutant (Cat.# M3682)
has been rendered RNase H minus by the introduction of a point mutation in the RNase H
domain of the enzyme. M-MLV Reverse Transcriptase, RNase Minus, Point Mutant, is
functionally equivalent to the well-known RNase H minus reverse transcriptase,
SuperScript™ II. Promega also offers M-MLV Reverse Transcriptase, RNase H Minus (Cat.# M5301).
This enzyme is similar to M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant, except
that the RNase H activity is eliminated by a deletion mutation in the RNase H domain of
the enzyme. M-MLV Reverse Transcriptase, RNase H Minus, is functionally equivalent to the
common RNase H minus reverse transcriptase, SuperScript™.
References
- Eun, H.-M. (1996) Enzymology Primer for Recombinant DNA Technology, Academic
Press, San Diego, California, 427.
- Gerard, G.F. and D’Alessio, J. (1993) In: Methods in Molecular Biology, Vol.
16: Enzymes of Molecular Biology, Burrell, M.M., ed., Humana Press, Totowa, New Jersey,
73.
- Enzyme Resource Guide, Vol. 1: Polymerases BR075A, Promega
Corporation.
- Kacian, D.L. (1977) Meth. Virol. 6, 143.
- Houts, G.E. et al. (1979) Reverse transcriptase from avian
myeloblastosis virus. J. Virol. 29, 517.
- Roth, M.J., Tanese, N. and Goff, S.P. (1985) Purification and characterization of
murine retroviral reverse transcriptase expressed in Escherichia coli. J. Biol. Chem.
260, 9326.
- Verma, I.M. (1974) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York,
87.
(a)The PCR process is covered by patents
issued and applicable in certain countries. Promega does not encourage or support the
unauthorized or unlicensed use of the PCR process.
SuperScript is a trademark of Life Technologies, Inc. |
