Reverse Transcription

Reverse transcription is the process of a double stranded cDNA synthesis on a single stranded RNA template. Initially found in retroviruses this mechanism further was determined also in several processes within eukaryotic and bacteria cells.

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The reaction is driven by an enzyme known as reverse transcriptase, revertase, or RNA-dependent DNA polymerase, and the final product is complementary DNA (c DNA). Since it was discovered, reverse transcription and corresponding enzymes from retroviruses have found extensive use in molecular cloning procedures, primarily for cDNA libraries creation. Combined with PCR (RT-PCR) reverse transcription allows detection of low RNA levels and therefore be an excellent tool for molecular diagnostics.

The History of Discovery

The phenomenon of reverse transcription was first described by Soviet scientists from the group of Sergey Gershenson in 1963 [1], but the works published in Russian and Ukrainian remained unnoticed. It was not until the early 1970s when the American teams headed by Howard Temin and David Baltimore independently rediscovered this machinery and isolated the reverse transcriptase enzymes (RT) from Murine leukemia virus and Rous sarcoma virus. In 1975 the discovery was awarded the Nobel Prize in Physiology and Medicine [2].

The process was called "reverse transcription", as it is the opposite of "normal" transcription, where the product (messenger RNA, etc.) is converted from the DNA template. It turned out that reverse transcription is characteristic of retroviruses — RNA viruses, such as HIV-1 (human immunodeficiency virus) or M-MLV (Moloney murine leukemia virus), which c DNA synthesized by RT integrates into the host genome and then can be expressed as "native" cellular DNA.

The reverse transcriptase enzyme activity has been later established in telomerase, the enzyme responsible for protection of telomeres within the eukaryotic chromosomes [3]. The enzyme has also been revealed to exist in bacteria where it is involved in the creation of DNA/RNA chimeric elements — multicopy single-stranded DNA (msDNA) [4].

Reverse Transcription Biology

The reverse transcription process in retroviruses is nowadays relatively well understood and described [5]. The template for synthesis is a viral genomic RNA that resembles an eukaryotic messenger RNA: it also contains 5'-cap and 3'-poly(a) tail.

The main stages of reverse transcription include:

  • Primer hybridization — lysyl-tRNA in nature, and short oligonucleotides (e. g. oligo(dT)) can be used in vitro;
  • Synthesis of complementary DNA first strand in 5'–3' direction downstream the primer;
  • The viral RNA component of the resulting hybrid is degraded by 3'–5' exonuclease (RNase H) domain of RT;
  • Second strand DNA synthesis: RTs have DNA-dependent DNA polymerase activity, or host enzyme is added to the process;
  • After primer leaves, the resulting DNA is ready for insertion into the chromosome, where it will now act as a part of the host genome.

Transcription and Reverse Transcription

The processes of "direct" and reverse transcription have a number of key differences in mechanisms. Despite the word "transcription" in its name, reverse transcription is much more similar to replication (DNA or viral RNA). The reverse transcriptase enzyme molecule has the so-called "right-hand" structure, typical for DNA polymerases and RNA-dependent RNA polymerases, but not for RNA polymerases responsible for DNA transcription [6]. Like similar enzymes, RT utilizes DNA sequence priming, and the final result of the reaction is a double-stranded molecule.

The table below summarizes the main differences between “direct” and reverse transcription.

ParameterTranscriptionReverse transcription
Enzyme DNA-dependent RNA polymerase Reverse transcriptase
Template DNA RNA
Reaction product mRNA, rRNA, tRNA, ribozymes and other (all RNA-world molecules excluding viral RNAs copied by replication) c DNA, telomere segments, msDNA
Primer None Lysyl tRNA
Additional enzyme activities involved Splicing, capping, polyadenylation DNA-dependent DNA polymerase activity, RNAse, integrase
Main biological mission Gene expression Genetic information storage within the host cell, gene rearrangement
Organisms Eukaryotes, prokaryotes, DNA-viruses Retroviruses, hepadnaviruses, eukaryotes (telomerase), prokaryotes
Application in bioengineering mRNA synthesis, protein expression cDNA synthesis, RT-PCR

Benefits and Applications of reverse transcription

Reverse transcription is widely used in molecular biology to study genes and various cellular and viral RNAs. The ability to obtain DNA copies from mRNA template (only encoding sequences, without introns and regulatory sequences) get us, first, the ability to use Sanger sequencing method to establish the genes identity and, second, the power to build cDNA libraries and to use these "gene records" for engineering tasks.

Additionally, labeled during the reverse transcription cDNA can be used in microarray and other assays requiring nucleic acid visualization (e.g., Southern blotting).

Combined with PCR (RT-PCR) reverse transcription allows the detection of a very low number of RNA copies in the sample, thus giving us an excellent tool for molecular diagnostics.

For this reason, many engineered RTs were produced with certain characteristics that differ from those in wild enzymes. For example, we can compare two enzymes, issued by Biolabmix, created on the basis of Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase, applicable for different experimental aims.

 RNAscribe RTM-MuLV–RHWild type M-MuLV RT [7]
Primer usage + +
RNAse H activity +
t°C optimum 50 – 55°C 42 – 45°C 37°C
t°C maximum 60°C 50°C 42°C
cDNA length up to 9000 b.p. up to 7000 b.p. about 8000 b.p.(?)

Such alterations as reduction of RNAse activity or rising the reaction temperature assist to improve the enzyme fidelity and the efficiency of full-length product formation, as well as allow it to work in the reverse transcriptase / PCR bundle and to process RNA molecules with complicated secondary structure.

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Additional facilities for studies connected to reverse transcription are ready-to-use kits for RT reactions: you can increase the productivity of your work, by increasing the accuracy of pipetting and reducing the errors in the reaction-related calculations. Moreover, these kits may help to normalize the conditions for producing multiple routine reactions and to provide relevant statistics.
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