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Reverse Transcriptases Overview

A major factor to consider in reverse transcription polymerase chain reaction (RT-PCR) is the choice of the appropriate reverse transcriptase.

Additional Reverse Transcriptases

Roche has developed and optimized a wide variety of conventional enzymes and enzyme blends. Each of these products has different enzymatic properties and one may be more suitable for a specific experiment than the other. The information given below outlines the individual properties of each enzyme and may help you find the optimum enzyme or enzyme blend for your specific application.

One-step RT-PCR - offers faster reactions, reduced risk of contamination, and improved sensitivity

  Tth DNA Polymerase Titan One Tube RT-PCR System Titan One Tube RT-PCR Kit
Composition Thermostable DNA polymerase with intrinsic reverse transcriptase activity. AMV reverse transcriptase and Expand High Fidelity Enzyme Blend.
Product Size Up to 1 kb Up to 6 kb
Priming Specific Specific
Reaction Temperature +55 to +70°C +45 to +60°C
RNase H Activity - ++
Ion Requirement Mn2+ Mg2+
Sensitivity - Total RNA + 1 pg to 1 µg
Sensitivity - mRNA + ++
Sensitivity - Viral RNA + +
Sensitivity - RNA Rich in Secondary Structures + +
Specificity + +
Fidelity Highly processive 5´-3´ polymerase, lacking 3´-5´ exonuclease or proofreading activity. +
Full-Length cDNA - ++
Incorporation of Modified Nucleotides Yes Yes
Carryover Prevention Yes No
Buffer System Both a PCR and RT-PCR buffer are included. A single reaction buffer is included.
Advantages • Resistant to prolonged incubation at
  high temperature (+95°C).
• No RNase H activity.
• Carryover prevention.
• Long RT-PCR products; up to 6 kb.
• High sensitivity for mRNA.

 

Two-step RT-PCR - offers maximum flexibility with respect to choice of primers, enzyme, temperature, and analysis of multiple transcripts

  Reverse Transcriptase
M-MuLV
Expand Reverse Transcriptase Reverse Transcriptase AMV First Strand cDNA Synthesis Kit cDNA Synthesis System
Product Size 10 kb 14 kb 12 kb
Priming Specific Oligo(dT) Random hexamer Specific Oligo(dT) Random hexamer Specific Oligo(dT) Random hexamer
Reaction Temperature +37°C +42°C (up to +50°C) +42°C (up to +60°C)
RNase H Activity + - ++
Sensitivity - Total RNA + ++ +
Sensitivity - mRNA + ++ ++
Sensitivity - Viral RNA + + +
Sensitivity - RNA Rich in Secondary Structures +(+) + +
Specificity Not determined + +
Fidelity + + +
Full-Length cDNA + +++ ++
Incorporation of Modified Nucleotides Yes Yes Yes
Carryover Prevention Not determined Not determined Not determined
Buffer System A single reaction buffer is included. Separate vials of DMSO, DTT, and Betain for optimization of reaction conditions are also included. A single reaction buffer is included. A separate vial of DTT for optimization of reaction conditions is also included. All necessary reagents are included.
Advantages • No endonuclease
  activity.
• Lower RNase H
  activity than AMV.
• cDNA fragments
  up to 14 kb.
• Full-length cDNA
  fragments.
• No RNase H
  activity.
• cDNA fragments up to 12 kb.
• Higher thermostability and
  specificity than M-MuLV.