Author Interviews, Genetic Research, Science, University Texas / 25.06.2016
Fixing An Evolutionary Omission: Adding Proof-Reading to Reverse
MedicalResearch.com Interview with:
Jared Ellefson, PhD
Postdoctoral fellow
University of Texas Austin's Center for Systems and Synthetic Biology
MedicalResearch.com: What is the background for this study? What are the main findings?
Response: Reverse transcriptases (RT) have revolutionized the field of biology - enabling the conversion of RNA into DNA. This initially allowed the cloning of mature messenger RNA into cDNA libraries (e.g. cloning human genes), but has since been finding a more modern role in high throughput RNA-seq which can accurately depict the physiological status of a cell. Despite its critical role, an inherent flaw exists in all known reverse transcriptases. They make many errors while copying RNA - due to the lack of an error-checking (proofreading) domain. Consequently, the errors produced in reverse transcription are propagated into RNA sequencing potentially leading to corrupted data.
The reason for the low fidelity of reverse transcriptases is due to their evolutionary heritage. All RTs are evolved from polymerase enzymes which lack the proofreading domain. This is in stark contrast to certain DNA polymerases which have extreme fidelity. The idea was, what if you could take a high fidelity DNA polymerase and transform it into a high fidelity RT. To do this we developed directed evolution techniques that would enrich these DNA polymerases for reverse transcriptase activity. After a monumental engineering effort, we were left with the world's first reverse transcriptase that could error-check during polymerization. We found that this increased the fidelity of RNA sequencing, in addition to a number of other interesting properties (for instance this single enzyme can do both reverse transcription and PCR).
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