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In-vitro Mutagenesis01:16

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

In vitro selection using modified or unnatural nucleotides.

Scott M Knudsen1, Michael P Robertson, Andrew D Ellington

  • 1University of Texas, Austin, Texas, USA.

Current Protocols in Nucleic Acid Chemistry
|April 23, 2008
PubMed
Summary
This summary is machine-generated.

Modified nucleotides enhance nucleic acid stability for in vitro selection. This guide offers protocols for preparing, verifying, and utilizing modified RNA pools for successful in vitro selection and amplification.

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Published on: March 16, 2011

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • In vitro selection is a powerful technique for isolating nucleic acids with desired functions.
  • Standard nucleic acid pools are susceptible to degradation by nucleases, limiting selection efficiency.
  • Modified nucleotides offer potential solutions to enhance stability and expand the utility of in vitro selection.

Purpose of the Study:

  • To provide comprehensive protocols for in vitro selection using modified nucleotides.
  • To guide researchers on the advantages and applications of modified nucleotides in nucleic acid selection.
  • To detail methods for preparing and validating modified RNA pools for in vitro selection.

Main Methods:

  • Preparation of modified RNA pools incorporating various modified nucleotides.
  • Verification of modified RNA pool integrity and suitability for in vitro selection using techniques like PAGE and mass spectrometry.
  • In vitro selection and amplification protocols tailored for modified nucleic acid pools.

Main Results:

  • Demonstration of increased nuclease resistance in modified nucleic acid pools compared to unmodified pools.
  • Successful isolation of functionally enriched pools using modified nucleotides.
  • Establishment of reliable protocols for handling and selecting modified nucleic acids.

Conclusions:

  • Modified nucleotides significantly improve the stability of nucleic acids during in vitro selection.
  • The provided protocols enable efficient preparation and application of modified RNA pools.
  • This approach expands the scope and success rate of in vitro selection for diverse applications.