Life Science

Code No. TRT-101 10,000U
*This product is not available in the US.

High Efficient Reverse Transcriptase
ReverTra Ace®

Description

ReverTra Ace® is a high efficient M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase that has been genetically modified to remove RNase H activity and increase reaction efficiency. It is the preferred enzyme for applications requiring full-length cDNAs and high product yields from total RNA, mRNA, rRNA, etc.

Features

  • RNase minus M-MLV RTase with improved performance
  • Enables the synthesis of longer cDNAs (≥ 14 kb) than the WT-enzyme
  • Exhibits excellent reaction efficiency at high temperatures.

Applications

  • cDNA synthesis
  • cDNA library construction
  • RT-PCR
  • 5’ RACE

Source

E. coli strain carrying a cloned, modified M-MLV reverse transcriptase gene.

Unit definition

One unit is defined as the amount of enzyme required to incorporate 1 nmole of dTTP into an acid-insoluble material in 10 min at 42ºC.

Storage condition

50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 10 mM DTT, 0.01% Nonidet® P-40, 50% Glycerol. Store at -20ºC

Components

ReverTra Ace® (100U/μl)
5 x Buffer

Typical PCR reaction setup

<cDNA synthesis for RT-PCR>
Component Amount
RNA
  Total RNA
  Poly (A)+ RNA		  
0.1-1 μg
50-500 ng		 <Reaction condition>

30ºC, 10 min**
42ºC, 20-60 min
99ºC, 5 min

** when using random primers
Primer
  Oligo (dT)
  Random
  Gene specific		  
5 pmoles
25 pmoles
5 pmoles
5 x Buffer 4 μl
10 mM dNTPs* 2 μl
(RNase inhibitor ) 20 U
Nuclease-free water X μl
Total reaction volume 20 μl
* This reagent is not supplied with this product.
<1st strand cDNA synthesis>
Component Amount
Poly (A)+ RNA 50-500 ng <Reaction condition>

42ºC, 30 min
99ºC, 5 min
Oligo (dT) primer 5 pmoles
5 x Buffer 4 μl
10 mM dNTPs* 2 μl
(RNase inhibitor ) 20 U
Nuclease-free water X μl
Total reaction volume 20 μl
* This reagent is not supplied with this product.

Application data

Example 1. Comparison of elongation capability of RNase H minus RTases at various temperatures

cDNAs were synthesized with oligo (dT)30 primers and 100 U enzyme/poly (A)+ RNA mixture (1.35-9.49 kb, 0.4 mg) as templates for 30 min at various temperatures. cDNAs were labeled with [32P-dCTP] during the reaction. The synthesized cDNAs were separated by 1% denatured agarose gel electrophoresis, and detected. The results suggested that ReverTra Ace® can elongate efficiently at 42-55ºC compared to other RNase H minus RTases from other companies.

Example 2. Comparison of cDNA synthesis efficiency by RT-PCR

G3PDH genes (500 bp) were amplified by PCR using cDNA templates that were synthesized with various RNase H minus RTases from G3PDH mRNA (102-105 copies/reaction). The RTase reaction was performed with specific reverse primers and 100 U enzyme at 42ºC for 20 min. The results indicated that ReverTra Ace® is suitable for RT-PCR amplifications that require sensitivity.

Example 3. Confirmation of the elongation capability of a long cDN

cDNA was synthesized by ReverTra Ace® using a specific primer for the 3’-end of dystrophin mRNA at 42ºC for 30 min. The 5’ region at a distance of 14 kb from the 3’ end of the dystrophin gene was amplified by PCR. The result indicated that ReverTra Ace® can elongate cDNA of ≥ 14 kb.

References

  1. K. Miyazaki, H. Miyamoto, D.K. Mercer, T. Hirase, J.C. Martin, Y. Kojima, H.J. Flint, Involvement of the multidomain regulatory protein XynR in positive control of xylanase gene expression in the ruminal anaerobe Prevotella bryantii B(1)4. J Bacteriol. 185: 2219-26 (2003)
  2. A. Nezu, A. Tanimura, T. Morita, K. Irie, T. Yajima, Y. Tojyo, Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells. Biochem J. 363: 59-66 (2002)
  3. S. Nakamura, A. Ikegami, Y. Matsumura, T. Nakanishi, K. Nomura, Molecular cloning and expression of the mannose/glucose specific lectin from Castanea crenata cotyledons. J Biochem. 13: 241-6. (2002)
  4. S. Atsumi, Y. Ikawa, H. Shiraishi, T. Inoue, Design and development of a catalytic ribonucleoprotein. EMBO J. 2: 5453-60 (2001)
  5. Y. Yabuta, K. Yoshimura, T. Takeda, S. Shigeoka, Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli. Plant Cell Physiol. 41: 666-75 (2000)
  6. S. Lee. Y. Takeda, H. Kawano, H. Hosoya, M. Nomoto, D. Fujimoto, N. Takahashi, K. Watanabe. Expression and regulation of a gene encoding neural recognition molecule NB-3 of the contactin/F3 subgroup in mouse brain. Gene, 245: 253-66 (2000)

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