sho-maxmima

Molecular evolution for maximum cDNA synthesis

Thermo Scientific™ Maxima™ Reverse Transcriptases were developed through molecular evolution, which enabled the introduction and selection of multiple favorable mutations in traditional M-MuLV reverse transcriptase that maximize performance in cDNA synthesis.

Maximize performance in RT-PCR and RT-qPCR

Using our proprietary technology for in vitro protein evolution, multiple mutations were introduced into traditional M-MuLV reverse transcriptase, and those that conferred favorable attributes were selected. These new features of Maxima Reverse Transcriptases dramatically improved enzymes' thermostability, robustness, and processivity and improved their performance in cDNA synthesis.

Maximize performance in RT-PCR and RT-qPCR video
How to perform gDNA removal and cDNA synthesis in a single tube

Advantages

Maxima Reverse Transcriptase enzymes outperform other reverse transcriptases over a wide temperature range. Their tolerance of high reaction temperatures allows efficient transcription of RNA regions with extensive secondary structure and helps improve primer specificity, resulting in high yields of full-length DNA.

High yields of cDNA over a broad temperature range

High yields of cDNA over a broad temperature range. cDNA synthesis incorporating a radioactive label, using 1 μg of Invitrogen™ Millennium™ RNA markers (poly(A)-tailed) with oligo(dT)18 primer, was performed with Maxima H Minus Reverse Transcriptase and compared to cDNA synthesis performed according to the manufacturers’ protocol with reverse transcriptases from other vendors. Reaction products were resolved on an alkaline agarose gel.

Designed with proprietary mutations for enhanced performance, Maxima H Minus Reverse Transcriptase is capable of full-length cDNA synthesis from very long RNA templates.

gel image showing amplification of targets up to 13.3 kb in two step RT-PCR.

  Amplification of long targets in two-step RT-PCR. Total RNA (1 μg) from mammalian cells was used in reverse transcription reaction replicates with Maxima H Minus Reverse Transcriptase and reverse transcriptases from other vendors, according to manufacturers’ recommendations. The resulting cDNA products were used as templates for PCR and the results were visualized on agarose gels. Only Maxima H Minus Reverse Transcriptase was able to generate very long (13.3 kb) products with high yields

Maxima Reverse Transcriptase is capable of reproducible cDNA synthesis from a wide range of template amounts, making it an ideal choice for RT-qPCR experiments. The premixed solutions of Thermo Scientific™ Maxima™ First Strand cDNA Synthesis kits further help improve reproducibility and save time during reaction setup

graph shows reproducible cDNA synthesis and low variability (<1% SD/Ct) with a wide range of starting RNA amounts

  Reproducible cDNA synthesis and low variability (<1% SD/Ct) with a wide range of starting RNA amounts. First-strand cDNA was synthesized from 100, 10, 1, 0.1, 0.01, and 0.001 ng of total RNA from mammalian cells using the Maxima First Strand cDNA Synthesis Kit in 16 replicate reactions each. Synthesized cDNA was used as a template in qPCR with Thermo Scientific™ Maxima™ SYBR™ Green/ROX™ qPCR Master Mix. The amplification plot illustrates good consistency across the range of starting material.

Maxima First Strand cDNA kits include double-strand specific DNase (dsDNase) that allows genomic DNA elimination followed by cDNA synthesis, without any intermediate sample purification steps. The dsDNase is engineered to remove contaminating genomic DNA in two minutes without damaging RNA quantity or quality, or degrading single-stranded DNA such as primers and probes. The dsDNase is easily inactivated by modest heat treatment (55°C). The subsequent genomic DNA-free RNA sample can be used directly in cDNA synthesis reactions therefore significantly simplifying the experimental workflow.

graph showing effective removal of genomic DNA using dsDNase

  Effective removal of genomic DNA using dsDNase. Two-step RT-qPCR analysis of the PBGD gene with (RT+) and without (RT-) reverse transcriptase was plotted for several cDNA synthesis kits. cDNA synthesis was performed from 0.2 ng total Jurkat RNA using the Maxima First Strand cDNA Synthesis Kit with dsDNase or kits from other vendors that offer gDNA removal. The orange, flat RT- plot for reactions using the Maxima kit indicates complete removal of contaminating gDNA, whereas RT- reactions using kits from other suppliers indicates amplification of residual gDNA.
 

RNase inhibitors are typically denatured at higher temperatures, releasing the RNases back into the reaction mix which can damage or degrade RNA. For RNA protection against RNases, all Maxima First Strand cDNA Synthesis kits include engineered, thermostable Thermo Scientific™ RiboLock™ RNase Inhibitor. The protein “locks” onto RNases and thus prevents RNA degradation at temperatures up to 55 °C. The high thermostability of RiboLock RNase Inhibitor is important for successful reverse transcription at higher reaction temperatures.

gel image RiboLock RNase Inhibitor efficiently protects RNA and inhibits up to 2 ng/20 μL of RNase A

  RiboLock RNase Inhibitor efficiently protects RNA and inhibits up to 2 ng/20 μL of RNase A. Aliquots (20 μL) of total human RNA (1 μg) were supplemented at 37°C with 20 U of RiboLock RNase Inhibitor and increasing amounts of RNase A.
M: Thermo Scientific™ RiboRuler™ High Range RNA Ladder, ready-to-use
C: Total human RNA
1: Total human RNA with RNase A
2–5: Total human RNA with RiboLock RNase Inhibitor and RNase A

RiboLock RNase Inibitor exhibits high thermostability. Aliquots (20 μL) of total human RNA (1 μg) were supplemented with 20 U of RiboLock RNase Inhibitor and 50 pg of RNase A and incubated at increasing temperatures.

 

gel image shows RiboLock RNase Inibitor exhibits high thermostability

  RiboLock RNase Inibitor exhibits high thermostability. Aliquots (20 μL) of total human RNA (1 μg) were supplemented with 20 U of RiboLock RNase Inhibitor and 50 pg of RNase A and incubated at increasing temperatures.
M: RiboRuler High Range RNA Ladder, ready-to-use
C: Total human RNA
1: Total human RNA with RNase A
2–6: Total human RNA with RiboLock RNase Inhibitor and RNase A

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 M-MuLV 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 M-MuLV 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 M-MuLV 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 molecular evolution

Understanding RNase H activity

Wild type M-MuLV 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.

RNase H activity

Reverse transcriptase enzymes

Product Included
Maxima H Minus Reverse Transcriptase, 200 U/µL Supplied with 5X RT Buffer (250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, 15 mM MgCl2, 50 mM DTT).
Maxima Reverse Transcriptase, 200 U/µL Supplied with 5X RT Buffer (250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, 15 mM MgCl2, 50 mM DTT).

cDNA synthesis kits

Product Included
Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase Kit 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 H Minus First Strand cDNA Synthesis Kit Kit 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, and nuclease-free water.
Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase Kit 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 First Strand cDNA Synthesis Kit for RT-qPCR Kit components: Maxima Enzyme Mix (Maxima Reverse Transcriptase and RiboLock RNase Inhibitor), 5X Reaction Mix (reaction buffer, dNTPs, Oligo (dT)18 and random hexamer primers), and nuclease-free water
Maxima H Minus Double-Stranded cDNA Synthesis Kit Kit 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 primers, water.