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Related Concept Videos

In vitro Mutagenesis01:16

In vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

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Related Experiment Video

Updated: May 8, 2026

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
05:46

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Published on: April 9, 2014

Natural diversity-guided catalytic-core chimerism engineers a rapid and inhibitor-tolerant reverse transcriptase.

Inês Fonseca Costa1,2,3, Vânia Ondina Fernandes1, Rita Silva Simões1,2,3

  • 1NZYtech - Genes & Enzymes, Campus do Lumiar, Building J, Lisbon, 1649-038, Portugal.

Journal of Biological Engineering
|May 7, 2026
PubMed
Summary

Researchers engineered a novel reverse transcriptase (RT) enzyme, chRT V18, by combining natural enzyme variants. This new RT enzyme offers enhanced speed, stability, and inhibitor resistance for molecular diagnostics.

Keywords:
Inhibitor resistantM-MuLV RTProtein chimeraProtein engineeringReverse transcriptaseThermostable RT

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Last Updated: May 8, 2026

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
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Published on: April 9, 2014

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Published on: March 25, 2020

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Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

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Area of Science:

  • Molecular Biology
  • Enzyme Engineering

Background:

  • Reverse transcriptases (RTs) are crucial for RNA-based technologies but face limitations in speed, stability, and inhibitor tolerance.
  • Traditional optimization methods struggle to integrate multiple desired traits into a single RT enzyme.

Purpose of the Study:

  • To develop a novel reverse transcriptase with improved performance characteristics by overcoming limitations of existing enzymes.
  • To engineer an RT enzyme that integrates enhanced catalytic speed, thermal stability, and inhibitor resistance.

Main Methods:

  • Applied catalytic-core recombination using natural sequence diversity to create chimeric RT variants.
  • Swapped a 405-residue polymerase domain into an M-MuLV scaffold to generate a library of chimeras.
  • Screened chimeras for improved performance criteria, including temperature compatibility and inhibitor resistance.

Main Results:

  • Identified chRT V18 as a lead candidate with consistent activity from 40-70°C.
  • chRT V18 enabled rapid (1-min) and linear cDNA synthesis at elevated temperatures, with strong inhibitor resistance.
  • Demonstrated high-temperature RT-LAMP at 69°C and improved performance in one-step RT-qPCR master mixes, reducing reaction times by 90%.

Conclusions:

  • Natural diversity-guided chimerism successfully created a rapid, inhibitor-tolerant RT with broad temperature compatibility and minute-scale reverse transcription.
  • Modular recombination of evolutionarily optimized domains is a viable strategy for engineering complex enzymes with multiple integrated traits.
  • The developed RT enzyme, chRT V18, offers significant advantages for molecular diagnostic applications, enabling faster results and improved accuracy.