The Continuous Evolution In Vitro Technique

Carolina Díaz Arenas1, Niles Lehman1

1 Portland State University, Portland, Oregon
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 9.7
DOI:  10.1002/0471142700.nc0907s40
Online Posting Date:  March, 2010
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Abstract

In vitro experimentation techniques were developed in response to the necessity of exploring new molecular structures and functions and to better understand evolutionary phenomena that shape organismal and molecular populations. The advancement of these techniques has allowed further exploration of more complicated evolutionary dynamics. One such technique is the continuous evolution in vitro (CE) method, to which this unit is devoted. The CE method is characterized by continuous cycles of amplification of RNA molecules that occur without much participation of the researcher. This feature allows us to evolve lineages in which the evolutionary phenomena occurring at the molecular level more closely mimic what happens in organismal populations in the present, or what may have happened in RNA populations during the RNA world stage of life. Curr. Protoc. Nucleic Acid Chem. 40:9.7.1‐9.7.16. © 2010 by John Wiley & Sons, Inc.

Keywords: evolution; in vitro; mutation; RNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Continuous Evolution In Vitro (CE) Experiments
  • Alternate Protocol 1: Continuous Evolution In Vitro (CE) Experiments Using a Microfluidics System
  • Support Protocol 1: Determining the Dilution and Amplification Scheme
  • Support Protocol 2: Preparation of Ligase Ribozymes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Continuous Evolution In Vitro (CE) Experiments

  Materials
  • Stock ligase ribozymes [50 µM] (prepared in advance; see protocol 4)
  • CE buffer (prepared in advance; see recipe)
  • NTP mix (see recipe)
  • DEPC‐treated and/or RNase‐free water
  • PE mix (see recipe)
  • Ice bucket
  • 600‐µL and 1.5‐mL tubes
  • Timers
  • Benchtop cooler
  • 37°C heating block
  • Quick‐spin minifuge (ISC BioExpress, cat. no. C1301)
  • Additional reagents and equipment for carrying out PCR (e.g., Kramer and Coen, ) and agarose gel electrophoresis (e.g., Voytas, )

Alternate Protocol 1: Continuous Evolution In Vitro (CE) Experiments Using a Microfluidics System

  • α‐32P‐ATP (10 µCi/µL)
  • 2× XC (bromphenol blue with no xylene cylenol added) acrylamide gel‐loading dye
  • 5% polyacrylamide/8 M urea gel
  • Phosphorimager
  • Additional reagents and equipment for carrying out denaturing PAGE (e.g., see appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
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