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2014 ; 106
(ä): 7.16.1-7.16.24
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Genome-Wide Fitness and Genetic Interactions Determined by Tn-seq, a
High-Throughput Massively Parallel Sequencing Method for Microorganisms
#MMPMID24733243
van Opijnen T
; Lazinski DW
; Camilli A
Curr Protoc Mol Biol
2014[Apr]; 106
(ä): 7.16.1-7.16.24
PMID24733243
show ga
The lagging annotation of bacterial genomes and the inherent genetic complexity
of many phenotypes is hindering the discovery of new drug targets and the
development of new antimicrobial agents and vaccines. This unit presents Tn-seq,
a method that has made it possible to quantitatively determine fitness for most
genes in a microorganism and to screen for quantitative genetic interactions on a
genome-wide scale and in a high-throughput fashion. Tn-seq can thus direct
studies on the annotation of genes and untangle complex phenotypes. The method is
based on the construction of a saturated transposon insertion library. After
library selection, changes in the frequency of each insertion mutant are
determined by sequencing flanking regions en masse. These changes are used to
calculate each mutant's fitness. The method was originally developed for the
Gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia
and meningitis, but has now been applied to several different microbial species.