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.

Fig. 1. Point mutation in M-MLV RTase

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 conditionn

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 reaction set up

<cDNA synthesis for RT-PCR>

Component Amount
RNA Total RNA
Poly (A)+ RNA
0.1-1 µg
50-500 ng
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.

<Reaction condition>

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

** when using random primers

<1st strand cDNA synthesis>

Component Amount
Poly (A)+ RNA 50-500 ng
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.

<Reaction condition>

42ºC, 30 min
99ºC, 5 min

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 cDNA

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

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  • 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)