add

Find an Article

Saturday, September 20, 2014

Reverse Transcriptases used in RT-PCR

Reverse Transcription is a process in which RNA is converted to cDNA (complementary DNA). The enzyme responsible for reverse transcription is called Reverse Transcriptase.

The Retroviral Reverse Transcriptases has 3 major activities:

  1. RNA-dependent DNA Polymerase. 
  2. Ribonuclease H (RNase H) activity. 
  3. DNA-depedent DNA Polymerase. 
Three forms of RNA-dependent DNA polymerases are mostly used in the invitro synthesis of complementary DNA from RNA template.

1. Mesophilic enzyme encoded by Avian Myeloblastosis Virus (AMV)

2. Moloney Strain of Murine Lukemia Virus (Mo-MLV)

3. Thermostable Tth Taq DNA Polymerase.

Avian Myeloblastosis Virus (AMV) Reverse Transcriptase

The enzyme requires an RNA or DNA template with RNA or DNA Primer have 3’ Hydroxyl group. The enzyme lacks 3’-5’ exonuclease activity, because of this is it prone to introduce error.

The enzyme encoded by AVM shown have high RNase H activity, digests RNA moiety of RNA DNA hyrid and can cleave at template near 3’ terminus of the growing DNA strand if the reverse transcriptase pause synthesis.

The high RNase H activity of AMV can suppress the cDNA yield and restricts its length.

Moloney Strain of Murine Lukemia Virus (Mo-MLV) Revrese Transcriptase
Similar to AMV, Moloney Strain of Murine Lukemia Virus (Mo-MLV) also requires an RNA or DNA template with RNA or DNA Primer have 3’ Hydroxyl group. The enzyme lacks 3’-5’ exonuclease activity, because of this is it prone to introduce error.

RNase H activity of Mo-MLV is weak compared to the AMV, so it’s a better choice for use in RT-PCR.

Mo-MLV RTase reaches maximum activity at 37dC, where as AMV RTase reaches maximum activity at 42dC.

Theremostable Tth DNA polymerase
Tth DNA polymerase is encoded by Thermus thermophilus and the enzyme posses reverse transcriptase activity in the presence of Mn2+. Tth DNA polymerase has the advantage that the same enzyme can be used in both reverse transcription and amplification in same reaction tube.

The main disadvantage of Tth DNA polymerase is that the average size of the cDNA synthesized is 1-2Kb. The use of Mn2+ can lower the fidelity of DNA synthesis.

Commercially available Modified Reverse Transcriptases
Wild type enzyme is modified to yield improved performance. This includes improved transcription rates and longer cDNA strand synthesis. The modified enzymes are active even at 50 dC.

List of Commercially avilable Reverse Transcriptases:
  • RevertAid Reverse Transcriptase, Thermo Scientific 
  • SuperScript® Reverse Transcriptase, Life technologies 
  • StrataScript® Reverse Transcritase, Stratagene 
  • M-MLV Reverse Transcriptase, Promega 
  • M-MLV Reverse Transcriptase, Sigma 
References:
Technical Resources : Invitrogen
Technical Resources : Sigma
Technical Resources : Promega
Technical Resources : Thermo Scientific
Molecular Cloning, A Laboratory Manual, Sambrook

All company and/or product names may be trade names, trademark's and/or registered trademark's of the respective owners with which they are associated.

Recent Posts