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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 
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.

Tuesday, September 2, 2014

DNA Regulatory Sequence Analysis / DNA Binding Protein Analysis

Gene regulation is a tightly controlled and regulated process. Gene regulation is complex process and involves various control checkpoints. Promoters, enhancers, DNA binding proteins plays major role in gene regulation.

         Transcription of gene is controlled / regulated by Promoter and enhancers.

         The activity of enhancers and promoters are controlled by Transcription factors (the DNA binding Proteins).

         The DNA binding proteins plays a major role in the gene regulation, which can be identified by the following methods.

Methods for Identifying DNA binding Proteins / Gene Regulation

Following are some of the methods used for identification and analysis of gene regulation

         DNA Foot printing – Identifies site at which the protein binds.

         Gel Shift Assay – Identifies DNA-Protein Complex.

         CAT Assay – Measures Transcriptional Activity.

DNA Foot Printing

In DNA Foot Printing method,

The 5’ end of cloned DNA fragment having the promoter or enhancer to be studied is radiolabeled. 

The labeled DNA fragments are then divided into 2 sets. The first is set is incubated with nuclear extract having DNA binding proteins and the second set is used as control without incubating. 

After the incubation, both sets are treated with nucleases. Nucleases cleave the DNA into smaller fragments. 

Nucleases cannot cleave the regions where the protein is bound. The nuclease treated products are run on gel and autoradiographed.

By comparing the migration patterns of control Sites at which the DNA binding proteins interacts can be identified. The X-ray film gives the DNA foot print.

Gel Shift Assay

In Gel Shift Assay method,

DNA migration under electric field is used to identify the DNA binding proteins interaction. The migration of DNA-Protein complex is much lesser than the DNA alone. The radiolabeled DNA fragments are incubated with nuclear extract having DNA binding proteins, after incubation DNA fragments are run on gel along with the control (Not Incubated).

 By looking at the autoradiographic X-ray film DNA-Protein complex can be identified.

CAT Assay

In CAT Assay method,

Reporter gene is cloned with the promoter to be studied. The promoter-reporter construct is introduced into the cell for analyzing the transcriptional level. Active Promoter will initiate transcription of the reporter gene and the level of product formation can be analyzed.

Mostly used reporter genes:

         CAT gene – Chloramphenicol Aceyl Transferase.

         Luciferase gene.

         Expression levels of these genes are used to analyze the promoter.


Lehniger, Biochemistry
J.D Watson, Recombinant DNA
Kuby, Immunology