Molecular Evolution for Maximum cDNA

Thermo Scientific Maxima Reverse Transcriptases are developed through molecular evolution to maximize performance in cDNA synthesis.

Download the brochure


Maximize performance in RT-PCR and RT-qPCR

Our proprietary technology for in vitro protein evolution has enabled the introduction and selection of multiple favorable mutations into the traditional MMuLV reverse transcriptase. This has dramatically improved the enzyme thermostability, robustness and synthesis rates. Maxima Reverse Transcriptase and Maxima H Minus Reverse Transcriptase show 50× increase in processivity and are considerably more resistant to reaction inhibitors such as guanidine and formamide compared to wild type enzymes.


Advantages

Very high thermostability, full activity throughout the reaction

Maxima Reverse Transcriptases feature high thermal stability, leading to improved enzyme performance. cDNA synthesis can be performed at higher temperatures (up to 65 °C) ensuring successful transcription of RNA with high levels of secondary structure. In addition, due to the high thermostability, Maxima Reverse Transcriptases maintain their full activity throughout the RT reaction producing higher cDNA yields.

Residual activity after incubation at elevated temperatures
RTs were incubated at 50ºC (left) or at 60ºC (right) in RT reaction mixture. Residual enzyme activity was determined in a standard activity assay at indicated time points.

Reduced effects of reaction inhibitors

As proven for DNA polymerases such as Thermo Scientific Phusion DNA Polymerase, increased processivity allows for greater resistance to reaction inhibitors. The same phenomenon can be seen with Maxima Reverse Transciptases. They are 50× more processive than wild-type MMuLV RT and show significantly improved resistance to contaminating inhibitors such as guanidine, ethanol and formamide.

Maxima H Minus Reverse Transcriptase and Maxima Reverse Transcriptase maintain their activity in the presence of guanidine and ethanol whereas enzyme III from vendor A and wild-type MMuLV RT activity was reduced or lost in the presence of reaction inhibitors.

20 kb RT-PCR product – unrivaled performance in long RT-PCR

Due to its high processivity and lack of RNase H activity,Maxima H Minus Reverse Transcriptase is capable of full-length cDNA synthesis from very long RNA transcripts. As shown below, only Maxima H Minus RT was able to generate 20 kb long RT-PCR product in two step RT-PCR.

 

Amplification of targets up to 20 kb in two step RT-PCR.
1µg of a total RNA from Jurkat cells (1 and 2) or a total RNA from mouse skeletal muscle (3 and 4) were used in a reverse transcription reaction with Maxima H Minus First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template in subsequent PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:

  1. 3.9kb – BCL2 (human B-cell CLL/lymphoma 2)
  2. 6.8kb – POLE (human polymerase)
  3. 13.3kb – Dmd (mouse dystrophin)
  4. 20.2kb – Neb (mouse nebulin)
Ideal for RT-qPCR – sensitive and reproducible quantification

The RT step is one of the greatest sources of variation in RT-qPCR. Ideally, the Cq value should correlate with the initial number of RNA targets. Therefore, the RT enzyme used must perform equally well with a wide range of template amounts. In addition, the RT enzyme should be resistant to the traces of contaminants that arise from the RNA purification step. Maxima Reverse Transcriptase is capable of reproducible cDNA synthesis from 1 pg up to 5 μg in the presence of common reaction inhibitors, which makes it superior choice for your RT-qPCR experiments. To further improve the reproducibility in RT-qPCR, you can select Maxima First Strand cDNA Synthesis Kit with components premixed into two reagent tubes: Maxima RT with ribonuclease inhibitor and the reaction buffer with dNTPs and primers. This composition saves time and reduces variation due to pipetting.

 Reproducible cDNA synthesis and low variability levels (<1% SD/Cq) with a wide range of starting RNA amounts. First strand cDNA was generated from 100 ng-1 pg of total Jurkat cell RNA with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (#K1641) 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.
 
Full length cDNA at widest temperature range

Maxima Reverse Transcriptase and Maxima H Minus Reverse Transcriptase maintain full activity at widest temperature range from 42 °C to 65 °C superseding all other MMuLV RT derivatives. The high thermostability leads to efficient transcription of RNA regions with high secondary structure and improves specificity when gene-specific primers are used.

High yields of cDNA over a broad temperature range.
cDNA synthesis incorporating radioactive label using 1 μg of Ambion RNA Millennium™ markers (polyA tailed) with oligo(dT)18 primer at different temperatures. Reaction products were resolved on alkaline agarose gel.

7.5 kb cDNA in 5 min – fastest RTs in the industry

Maxima Reverse Transcriptase and Maxima H Minus Reverse Transcriptase were developed through in vitro evolution which resulted in improved enzyme processivity and speed. They are the only RT enzymes capable of synthesizing 7.5 kb long cDNA in just 5 min. As shown in the adjacent image, Maxima Reverse Transcriptases allow for the first strand cDNA synthesis reaction to be completed in 5-30 min with high yields. In contrast, the RT enzymes from other vendors deliver low or no yields.

 

 

High cDNA synthesis rate.
Synthesis of cDNA was performed for 5, 15, 30 and 60 minutes with different RTs at their optimal temperatures using 1 μg of 7.5 kb RNA transcript as a template. Reaction products were analyzed on a 1% alkaline agarose gel.


Understanding molecular evolution

The idea of using rational design criteria to improve enzyme performance is limited by our knowledge of polymerase fine structure and function. This limitation can be overcome by mimicking nature and using directed evolution for improving enzyme properties.

Our proprietary technique1, compartmentalized ribosome display (CRD), allows for fast and efficient in vitro evolution of reverse transcriptases. The technique has enabled the introduction and selection of multiple favorable mutations into wild-type MMuLV RT resulting in new, highly thermostable and processive reverse transcriptases that supersede their wild-type counterparts.

Molecular evolution by CRD technique comprises three steps. First, an mRNA library based on wild-type MMuLV RT gene is created by random mutagenesis. Next, the mRNA library is translated in vitro to proteins that are associated with their mRNA progenitors. Then, the protein-mRNA complexes are placed into RT reaction mixture and emulsified yielding compartments containing one protein-mRNA complex each. Finally, the temperature is increased to create a selective pressure under which only the improved mutants survive and produce full-length cDNA. By combining the best-performing mutations, highly processive MMuLV RT mutants capable of the full-length cDNA synthesis at high temperatures have been constructed.

1. Baranauskas et al. (2012). "Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants". PEDS. doi: 10.1093/protein/gzs034



Understanding RNase H activity

Wild type MMuLV RT possesses an RNA-dependent and DNA-dependent polymerase activity as well as RNase H activity. RNase H activity degrades RNA from RNA-DNA duplexes to allow efficient synthesis of dsDNA. However, with long mRNA templates, RNA may be degraded prematurely resulting in truncated cDNA. Hence, it is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning.

In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications, because they enhance the melting of RNA-DNA duplex during the first cycles of PCR.

Overview


Maximize performance in RT-PCR and RT-qPCR

Our proprietary technology for in vitro protein evolution has enabled the introduction and selection of multiple favorable mutations into the traditional MMuLV reverse transcriptase. This has dramatically improved the enzyme thermostability, robustness and synthesis rates. Maxima Reverse Transcriptase and Maxima H Minus Reverse Transcriptase show 50× increase in processivity and are considerably more resistant to reaction inhibitors such as guanidine and formamide compared to wild type enzymes.


Advantages

Very high thermostability, full activity throughout the reaction

Maxima Reverse Transcriptases feature high thermal stability, leading to improved enzyme performance. cDNA synthesis can be performed at higher temperatures (up to 65 °C) ensuring successful transcription of RNA with high levels of secondary structure. In addition, due to the high thermostability, Maxima Reverse Transcriptases maintain their full activity throughout the RT reaction producing higher cDNA yields.

Residual activity after incubation at elevated temperatures
RTs were incubated at 50ºC (left) or at 60ºC (right) in RT reaction mixture. Residual enzyme activity was determined in a standard activity assay at indicated time points.

Reduced effects of reaction inhibitors

As proven for DNA polymerases such as Thermo Scientific Phusion DNA Polymerase, increased processivity allows for greater resistance to reaction inhibitors. The same phenomenon can be seen with Maxima Reverse Transciptases. They are 50× more processive than wild-type MMuLV RT and show significantly improved resistance to contaminating inhibitors such as guanidine, ethanol and formamide.

Maxima H Minus Reverse Transcriptase and Maxima Reverse Transcriptase maintain their activity in the presence of guanidine and ethanol whereas enzyme III from vendor A and wild-type MMuLV RT activity was reduced or lost in the presence of reaction inhibitors.

20 kb RT-PCR product – unrivaled performance in long RT-PCR

Due to its high processivity and lack of RNase H activity,Maxima H Minus Reverse Transcriptase is capable of full-length cDNA synthesis from very long RNA transcripts. As shown below, only Maxima H Minus RT was able to generate 20 kb long RT-PCR product in two step RT-PCR.

 

Amplification of targets up to 20 kb in two step RT-PCR.
1µg of a total RNA from Jurkat cells (1 and 2) or a total RNA from mouse skeletal muscle (3 and 4) were used in a reverse transcription reaction with Maxima H Minus First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template in subsequent PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:

  1. 3.9kb – BCL2 (human B-cell CLL/lymphoma 2)
  2. 6.8kb – POLE (human polymerase)
  3. 13.3kb – Dmd (mouse dystrophin)
  4. 20.2kb – Neb (mouse nebulin)
Ideal for RT-qPCR – sensitive and reproducible quantification

The RT step is one of the greatest sources of variation in RT-qPCR. Ideally, the Cq value should correlate with the initial number of RNA targets. Therefore, the RT enzyme used must perform equally well with a wide range of template amounts. In addition, the RT enzyme should be resistant to the traces of contaminants that arise from the RNA purification step. Maxima Reverse Transcriptase is capable of reproducible cDNA synthesis from 1 pg up to 5 μg in the presence of common reaction inhibitors, which makes it superior choice for your RT-qPCR experiments. To further improve the reproducibility in RT-qPCR, you can select Maxima First Strand cDNA Synthesis Kit with components premixed into two reagent tubes: Maxima RT with ribonuclease inhibitor and the reaction buffer with dNTPs and primers. This composition saves time and reduces variation due to pipetting.

 Reproducible cDNA synthesis and low variability levels (<1% SD/Cq) with a wide range of starting RNA amounts. First strand cDNA was generated from 100 ng-1 pg of total Jurkat cell RNA with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (#K1641) 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.
 
Full length cDNA at widest temperature range

Maxima Reverse Transcriptase and Maxima H Minus Reverse Transcriptase maintain full activity at widest temperature range from 42 °C to 65 °C superseding all other MMuLV RT derivatives. The high thermostability leads to efficient transcription of RNA regions with high secondary structure and improves specificity when gene-specific primers are used.

High yields of cDNA over a broad temperature range.
cDNA synthesis incorporating radioactive label using 1 μg of Ambion RNA Millennium™ markers (polyA tailed) with oligo(dT)18 primer at different temperatures. Reaction products were resolved on alkaline agarose gel.

7.5 kb cDNA in 5 min – fastest RTs in the industry

Maxima Reverse Transcriptase and Maxima H Minus Reverse Transcriptase were developed through in vitro evolution which resulted in improved enzyme processivity and speed. They are the only RT enzymes capable of synthesizing 7.5 kb long cDNA in just 5 min. As shown in the adjacent image, Maxima Reverse Transcriptases allow for the first strand cDNA synthesis reaction to be completed in 5-30 min with high yields. In contrast, the RT enzymes from other vendors deliver low or no yields.

 

 

High cDNA synthesis rate.
Synthesis of cDNA was performed for 5, 15, 30 and 60 minutes with different RTs at their optimal temperatures using 1 μg of 7.5 kb RNA transcript as a template. Reaction products were analyzed on a 1% alkaline agarose gel.

Molecular evolution


Understanding molecular evolution

The idea of using rational design criteria to improve enzyme performance is limited by our knowledge of polymerase fine structure and function. This limitation can be overcome by mimicking nature and using directed evolution for improving enzyme properties.

Our proprietary technique1, compartmentalized ribosome display (CRD), allows for fast and efficient in vitro evolution of reverse transcriptases. The technique has enabled the introduction and selection of multiple favorable mutations into wild-type MMuLV RT resulting in new, highly thermostable and processive reverse transcriptases that supersede their wild-type counterparts.

Molecular evolution by CRD technique comprises three steps. First, an mRNA library based on wild-type MMuLV RT gene is created by random mutagenesis. Next, the mRNA library is translated in vitro to proteins that are associated with their mRNA progenitors. Then, the protein-mRNA complexes are placed into RT reaction mixture and emulsified yielding compartments containing one protein-mRNA complex each. Finally, the temperature is increased to create a selective pressure under which only the improved mutants survive and produce full-length cDNA. By combining the best-performing mutations, highly processive MMuLV RT mutants capable of the full-length cDNA synthesis at high temperatures have been constructed.

1. Baranauskas et al. (2012). "Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants". PEDS. doi: 10.1093/protein/gzs034


RNase H activity


Understanding RNase H activity

Wild type MMuLV RT possesses an RNA-dependent and DNA-dependent polymerase activity as well as RNase H activity. RNase H activity degrades RNA from RNA-DNA duplexes to allow efficient synthesis of dsDNA. However, with long mRNA templates, RNA may be degraded prematurely resulting in truncated cDNA. Hence, it is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning.

In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications, because they enhance the melting of RNA-DNA duplex during the first cycles of PCR.

Products - RT enzyme selection

Selection of Thermo Scientific reverse transcriptases (RTs) include both the advanced enzymes obtained through in vitro evolution of M-MuLV RT as well as the wild-type RT enzymes, offering a range of RT enzymes from routine to enhanced performance.

In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications, because they enhance the melting of RNA-DNA duplex during the first cycles of PCR.

ProductIncluded
Maxima H Minus First Strand cDNA Synthesis Kit with dsDNaseKit components: Maxima H Minus Enzyme Mix (Maxima H Minus Reverse Transcriptase and RiboLock RNase Inhibitor), Oligo(dT)18 and Random hexamer primers, 5X RT Buffer, dNTP Mix, dsDNase, 10X dsDNase Buffer and nuclease-free water.
Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNaseKit components: Maxima Enzyme Mix (Maxima Reverse Transcriptase and RiboLock RNase Inhibitor), 5X Reaction Mix (reaction buffer, dNTPs, oligo (dT)18 and random hexamer primers), dsDNase, 10X dsDNase Buffer, nuclease-free water
Maxima H Minus Reverse Transcriptase, 200 U/µlSupplied with 5X RT Buffer
Maxima H Minus First Strand cDNA Synthesis Kit*Kit components: Maxima H Minus Enzyme Mix (Maxima H Minus Reverse Transcriptase and RiboLock RNase Inhibitor), 5X RT Buffer, dNTP Mix, Oligo-(dT)18 Primer, Random Hexamer Primer and Water (nuclease-free).
Maxima H Minus Double-Stranded cDNA Synthesis KitKit components: First Strand Enzyme Mix , 4X First Strand Reaction Mix , Second Strand Enzyme Mix , 5X Second Strand Reaction Mix , 0.5 M EDTA, RNase I, Control RNA, Oligo(dT)18 Primer, Random Hexamer Primer, Water.
Maxima Reverse Transcriptase, 200 U/µlSupplied with 5X RT Buffer
Maxima First Strand cDNA Synthesis Kit for RT-qPCRKit components: Maxima Enzyme Mix (Maxima Reverse Transcriptase and RiboLock RNase Inhibitor), 5X Reaction Mix (reaction buffer, dNTPs, Oligo-(dT)18 Primer and Random Hexamer Primers) and Water (nuclease-free).
RevertAid H Minus Reverse Transcriptase, 200 U/µlSupplied with 5X RT Buffer
RevertAid H Minus First Strand cDNA Synthesis KitKit components: RevertAid H Minus Reverse Transcriptase, RiboLock RNase Inhibitor, 5X RT Buffer, dNTP Mix, Oligo-(dT)18 Primer, Random Hexamer Primer and Water (nuclease-free).
RevertAid Reverse Transcriptase, 200 U/µlSupplied with 5X RT Buffer
RevertAid First Strand cDNA Synthesis KitKit components: RevertAid Reverse Transcriptase, RiboLock RNase Inhibitor, 5X RT Buffer, 10 mM dNTP Mix, Oligo-(dT)18 Primer, Random Hexamer Primer and Water (nuclease-free).
Oligo (dT)18 PrimerRandom Hexamer Primer