Abstract
Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species.
Highlights
Simple sequence repeat (SSR) tracts are highly mutable sequences due to the potential for slip strand mis-pairing during DNA replication [1]
We have developed a Perl script (PSAnalyse) that will accept this output file of PeakScannerTM and analyse each sample to determine the fragment length, peak height, repeat number and expression state of the major peak for each of the 28 phase-variable loci of C. jejuni strain NCTC11168
The crucial features of the primer design for the multiplex PCR assay were:- high specificity of at least one primer within a pair for the target locus; and ensuring that no two PCR products had both the same or similar size (
Summary
Simple sequence repeat (SSR) tracts are highly mutable sequences due to the potential for slip strand mis-pairing during DNA replication [1]. A multiplex PCR and fragment analysis assay was previously developed for six of the polyG/polyC tracts of C. jejuni strain NCTC11168 and used to determine expression states for six genes in samples from in vitro and in vivo passage experiments [8]. Some of the phase-variable loci contain identical sequences in and around the repeat tracts necessitating development of locus-specific primers and careful design of multiplex PCR reactions.
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