Elegant Experiments that Changed the World

Crystallographic structure of HIV reversed transcriptase. Wikimedia Commons

Crystallographic structure of HIV reverse transcriptase. Wikimedia Commons

Today, reverse transcriptases are commonplace molecular biology tools, easy to obtain and routinely used in labs for everyday cloning and gene expression analysis experiments. Reverse transcriptase inhibitors have also found widespread use as antiviral drugs in the treatment of retroviral infections.

It’s easy to forget that the existence of reverse transcriptase activity—the ability to convert an RNA template into DNA—was once a revolutionary notion not easily accepted by the scientific community. The idea that RNA could be the template for DNA synthesis challenged the “DNA–>RNA–> Protein” central dogma of molecular biology.

The foundational studies that proved the existence of a reverse transcriptase activity in RNA tumor viruses were described in two papers published back-to-back in Nature in June, 1970. Two of the authors of these studies, Howard Temin of the University of Wisconsin and David Baltimore of the Massachusetts Institute of Technology, were awarded a Nobel Prize for their work in 1975.

In appreciation of the significance of these papers, the editorial introduction published in Nature at the time states:

This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can be inverted.

Before these papers were published, it was known that successful infection of cells by RNA tumor viruses required DNA synthesis. Formation of virions could be inhibited by Actinomycin D—an inhibitor of DNA-dependent RNA polymerase—so it was known that synthesis of viral RNA from a DNA template was part of the viral life cycle. The existence of an intracellular DNA viral genome was therefore indicated, and had been postulated by Temin in the mid 1960’s. However, proof of the mechanism whereby this DNA template was generated from the RNA genome of the infecting virus remained elusive. Continue reading

Dealing with PCR Inhibitors

InhibitionThe polymerase chain reaction (PCR) has revolutionized modern biology as a quick and easy way to generate amazing amounts of genomic data. However, when PCR doesn’t work, it can be frustrating. At these times, PCR and reverse transcription PCR (RT-PCR) inhibitors seem to be everywhere: They lie dormant in your starting material and can co-purify with the template of interest, and they can be introduced during sample handling or reaction setup. The effects of these inhibitors can range from partial inhibition and underestimation of the target nucleic acid amount to complete amplification failure. What is a scientist to do?

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Choosing the Right Reverse Transcriptase for Your Project

There are a lot of choices when it comes to reverse transcriptases.  Choosing the correct one for your cDNA synthesis and RT-PCR project is important.    Here are a few questions that will lead you to right RT for your application: Continue reading

The Evolution of Radical Notions in Science

HIV life cycleWhen I was in school and first learning about science, I was enthralled by how research was a quest to solve a problem or answer a question. In my teenage years, I refined my interest in a science career by deciding to become a biochemist and cure cancer. As I progressed through college, I realized this was too broad an endeavor for one person (i.e., the mechanism of carcinogenesis had vast and variable origins), and that biology was built on the knowledge, research and hypotheses of many scientists working on a singular subject. Continue reading