Abstract
We introduce a system for experimental evolution consisting of populations of short oligonucleotides (Oli populations) evolving in a modified quantitative polymerase chain reaction (qPCR). It is tractable at the genetic, genomic, phenotypic and fitness levels. The Oli system uses DNA hairpins designed to form structures that self-prime under defined conditions. Selection acts on the phenotype of self-priming, after which differences in fitness are amplified and quantified using qPCR. We outline the methodological and bioinformatics tools for the Oli system here and demonstrate that it can be used as a conventional experimental evolution model system by test-driving it in an experiment investigating adaptive evolution under different rates of environmental change.
Highlights
We are still unable to predict or interpret most of the genetic variation that occurs during laboratory selection experiments, even in our best-studied model organisms evolved in monocultures in simple environments, and many of our insights stem from case studies of exceptions rather than systematic surveys (Barrick et al, 2009)
It would be useful to know the distributions of fitness effects of beneficial mutations, of fixed mutations and of epistatic interactions between fixed mutations
We present a new in vitro system based on a modified quantitative polymerase chain reaction for carrying out laboratory selection experia 2013 THE AUTHORS
Summary
The unextended Oli molecule is a 107-bp oligonucleotide with the sequence 5′-AAGCAGTGGGagaCCGAGTTAtC CAaGtataGAGccgaCCccaacaaCAGCAGGCTGCTcccattaac ccCAGGCTCAGATCTGggTCTAtACttcCttcGGTCTCC-3′. PCRs were set up with a known number of starting molecules and subjected to a single round of selection (steps up to but not including restriction digestion) at self-priming temperatures from 45 to 65 °C followed by the addition of primers and SYBR green, 35 cycles of standard qPCR. We modified the protocol by using 30 lL sample added to 190 lL working solution and a finer-scale linear standard curve (0–1.0 ng/lL DNA), which produced good results, as verified by measuring known concentrations of the control Oli molecules The agreement between this measurement, which is based on the total amount of DNA present, and the initial number of molecules calculated by qPCR was used to calculate the proportion of the initial population that was able to fold and self-prime. The first sample (SC02) had been sequenced and reads randomly sampling (e.g. at 10 000, 20 000, 30 000 reads) to see at what read numbers new novel sequences stop appearing, to obtain an estimate the number reads required to maintain the same numbers of jMOTU clusters, the remaining fifteen samples were sequenced to obtain at least this number of reads
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