29Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_3, © Springer Science+Business Media, LLC 2018
Chapter 3
Method for Detecting and Studying Genome-Wide
Mutations in Single Living Cells in Real Time
Marina Elez, Lydia Robert, and Ivan Matic
Abstract
DNA sequencing and fluctuation test have been choice methods for studying DNA mutations for decades.
Although invaluable tools allowing many important discoveries on mutations, they are both highly influ-
enced by fitness effects of mutations, and therefore suffer several limits. Fluctuation test is for example
limited to mutations that produce an identifiable phenotype, which is the minority of all generated muta-
tions. Genome-wide extrapolations using this method are therefore difficult. DNA sequencing detects
almost all DNA mutations in population of cells. However, the obtained population mutation spectrum is
biased because of the fitness effects of newly generated mutations. For example, mutations that affect fit-
ness strongly and negatively are underrepresented, while those with a strong positive effect are overrepre-
sented. Single-cell genome sequencing can solve this problem. However, sufficient amount of DNA for
this approach is obtained by massive whole-genome amplification, which produces many artifacts.
We describe the first direct method for monitoring genome-wide mutations in living cells indepen-
dently of their effect on fitness. This method is based on the following three facts. First, DNA replication
errors are the major source of DNA mutations. Second, these errors are the target for an evolutionarily
conserved mismatch repair (MMR) system, which repairs the vast majority of replication errors. Third, we
recently showed that the fluorescently labeled MMR protein MutL forms fluorescent foci on unrepaired
replication errors. If not repaired, the new round of DNA synthesis fixes these errors in the genome per-
manently, i.e., they become mutations. Therefore, visualizing foci of the fluorescently tagged MutL in
individual living cells allows detecting mutations as they appear, before the expression of the phenotype.
Key words Mutation, Single-cell, Real-time, Genomic, Microscopy, MutL, Mismatch repair1 Introduction
Mutations are the raw material of evolution because they are the
ultimate source of all genetic variation. Newly arisen mutations can
have very different impact on the fitness of the organism, ranging
from deleterious through neutral to beneficial. Quantifying when
and how different mutations occur allows understanding and pre-
dicting the evolution of organisms. In bacteria, these parameters
are for example useful for predicting the rapidity with which they
can evolve different capacities, such as antibiotic resistance and