Introduction
Reverse transcription is the synthesis of DNA from an RNA template. This process is driven by RNA-dependent DNA polymerases, also known as reverse transcriptases. Reverse transcriptases occur naturally in both prokaryotic and eukaryotic organisms, as well as in retroviruses.
Reverse Transcriptase
Reverse transcriptases (RTs) are RNA-dependent DNA polymerases, a group of enzymes that play a unique role in the flow of genetic information. These enzymes enable the reverse transcription reaction and have been widely used by researchers in a variety of molecular biology applications since their discovery.
The endogenous properties of reverse transcriptases can be exploited and modulated for successful cDNA-based experiments. In addition to opening up the research into their native roles, including genetic diversity and retroviral replication, reverse transcriptases prove to be important tools for molecular biologists for various applications like gene expression analysis and cDNA sequencing.
The discovery of reverse transcriptase
The original central dogma of molecular biology held that DNA was transcribed to RNA, which in turn was translated into protein. However, this concept was challenged in the 1970s when two scientific teams, one led by Howard Temin at the University of Wisconsin and the other led by David Baltimore at MIT, independently identified new enzymes associated with replication of RNA viruses called retroviruses].
These enzymes, called reverse transcriptases, convert the viral RNA into a complementary DNA (cDNA) molecule, which then integrates into the host’s genome. In 1975, Temin and Baltimore received the Nobel Prize in Physiology or Medicine (shared with Renato Dulbecco for related work on tumor-inducing viruses) for their pioneering work in identifying reverse transcriptases.
The prevalence of reverse transcriptase in nature
Reverse transcriptases have been identified in many organisms, including bacteria, animals, and plants, as well as viruses. The natural role of reverse transcriptase is to convert RNA sequences to cDNA sequences that are capable of being inserted into different areas of the genome. In this manner, reverse transcription contributes to:
#Propagation of retroviruses—e.g., human immunodeficiency virus (HIV), Moloney murine leukemia virus (M-MuLV), and avian myeloblastosis virus (AMV).
#Genetic diversity in eukaryotes via mobile transposable elements called retrotransposons.
#Replication of chromosomal ends called telomeres.
#Synthesis of extrachromosomal DNA/RNA chimeric elements called multicopy single-stranded DNA (msDNA) in bacteria.
Applications of Reverse transcription
While reverse transcriptases have functional roles in biological systems, they also serve as important tools for studying RNA populations. In molecular biology, reverse transcriptases were first used to produce cDNA to build libraries. cDNA libraries contain DNA copies of mRNA from cells and tissues and are used to gain an understanding of actively expressed genes and their functions at a specific time point.
Although the creation of cDNA libraries was an important step forward in characterizing expressed genes, challenges remained for the study of low-abundance RNAs.
These were subsequently addressed with the development of the polymerase chain reaction (PCR), a technique to amplify small amounts of genetic material. Reverse transcription combined with PCR, or reverse transcription PCR (RT-PCR), allows detection of RNA even at very low levels of gene expression and paves the way for detection of circulating RNA, RNA viruses, and cancerous gene fusions in molecular diagnostics.
In addition, cDNAs serve as templates in applications such as microarray and RNA sequencing to characterize unknown RNAs in a high-throughput manner.
