New Maxima™ Reverse Transcriptase - High Yields of cDNA up to 20 kb

Features

• High yields of full-length cDNA up to 20 kb.
• Active up to 60°C.
• Thermostable – 90% active after incubation at 50°C for 60 minutes.
• Efficient – complete cDNA synthesis in 15-30 minutes.
• High sensitivity – reproducible cDNA synthesis from a wide range of starting RNA quantities (0.01 pg – 5 μg).
• Incorporates modified nucleotides.

Applications

• Two step RT-PCR.
• Two step RT-qPCR.
• First strand cDNA synthesis.
• Construction of full length cDNA libraries.
• DNA labeling.
• Primer extension.

Description

Maxima™ Reverse Transcriptase (RT) was developed by Fermentas through in vitro evolution of M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity as well as RNase H activity. The engineered enzyme features dramatically improved thermostability, robustness and increased synthesis rate compared to wild type M-MuLV RT. 

Maxima™ Reverse Transcriptase is capable of reproducible cDNA synthesis from a wide range of input total RNA amounts (from 0.01 pg to 5 μg) at elevated temperatures (50-60°C), which makes this enzyme an ideal tool for two step RT-qPCR (see Fig.5).

Due to its high thermostability (see Fig.1), the enzyme maintains full activity during the entire reverse transcription reaction, generates the highest yields of cDNA (see Fig.3) and is able to synthesize even very long RNA transcripts up to 20 kb (see Fig.2). The reaction temperature can be increased up to 60°C for efficient transcription of RNA regions with a high secondary structure or to improve specificity using gene specific primers. Due to its increased synthesis rate, the reverse transcriptase reaction can be completed in 15-30 min (see Fig.4).

Source

E.coli cells carrying an engineered pol gene fragment of Moloney Murine Leukemia Virus.

Definition of Activity Unit

One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 37°C. 

Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml [3H]-dTTP, 0.4 mM polyA•oligo(dT)12-18.

Storage Buffer

The enzyme is supplied in:

50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100 and 50% (v/v) glycerol.

5X RT Buffer

250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, 15 mM MgCl2, 50 mM DTT.

Quality Control

The absence of endodeoxyribonucleases, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests. Functionally tested in first strand cDNA synthesis.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate, pyrophosphate and polyamines.
• Inactivated by heating at 85°C for 5 min.

Figure 1. High thermostability of Maxima™ Reverse Transcriptase at 50°C.

Reverse transcriptases were incubated in 1X reaction buffer. At the indicated time points (5-240 minutes), enzyme activity was determined in a standard activity assay.

Figure 2. Amplification of targets up to 20 kb in two step RT-PCR.

1 µg of total RNA from Jurkat cells (1 and 2) or total RNA from mouse (3 and 4) were used in a reverse transcription reaction with Maxima™ Reverse transcriptase and other RTs in the optimal conditions for each enzyme. Synthesized cDNA was used as a template in PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:

1 – 6.8 kb Pole (human polymerase)
2 – 9.4 kb FBN1 (human fibrillin)
3 – 13.3 kb mouse distrophine
4 – 20.0 kb mouse nebuline
M – GeneRuler™ 1 kb Plus DNA Ladder (#SM1331)

Figure 3. High yields of cDNA over a broad temperature range.

Synthesis of 33P-labeled cDNA was performed using 200 units of each reverse transcriptase, 1 µg of Ambion RNA Millennium™ markers, and oligo(dT)18 primer at various temperatures. Reaction products were analyzed on a 1% alkaline agarose gel.

Figure 4. High cDNA synthesis rate at 50°C.

Synthesis of cDNA was performed at 50°C for 5, 15, 30 and 60 minutes using 1 µg of 7 kb RNA transcript as a template. Reaction products were analyzed on a 1% alkaline agarose gel. Only Maxima™ RT was able to complete synthesis of 7 kb transcript in 5 min.

Figure 5. Reproducible RT-qPCR results using Maxima™ Reverse Transcriptase.

100 ng-1 pg of total Jurkat cell RNA and a mix of Oligo(dT)18 and random hexamer primers were used with Maxima™ RT in 16 replicate reactions. Synthesized cDNA was used as a template in subsequent qPCR with Maxima™ SYBR Green/ROX qPCR Master Mix (#K0221) on the ABI 7500 Real-Time PCR instrument. Parallel reactions with Maxima™ RT demonstrate reproducible cDNA synthesis and low variability levels (<1% SD/Cq) with a wide range of starting RNA amounts.

Courtesy of Fermentas