Abstract
Fragment analysis (or fragment length analysis) is a PCR-based method which allows quantification of the size and proportion of a DNA repeat tract length of a phase-variable region. Primers are labeled with a fluorescent dye, the resulting amplicons are processed by capillary electrophoresis, and results are analyzed for amplicon size and proportion by associated software (such as Peakscanner). Here we describe the process of designing primers and controls to screen for the number of repeats in a polymeric tract of a phase-variable gene in Neisseria meningitidis (the DNA methyltransferase ModA is used as an example, but this method can be applied to other phase-variable genes).
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References
Livorsi DJ, Stenehjem E, Stephens DS (2011) Virulence factors of gram-negative bacteria in sepsis with a focus on Neisseria meningitidis. Contrib Microbiol 17:31–47
Rotman E, Seifert HS (2014) The genetics of Neisseria species. Annu Rev Genet 48:405–431
Srikhanta YN, Fox KL, Jennings MP (2010) The phasevarion: phase variation of type III DNA methyltransferases controls coordinated switching in multiple genes. Nat Rev Microbiol 8(3):196–206
Saunders NJ, Jeffries AC, Peden JF et al (2000) Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58. Mol Microbiol 37(1):207–215
Peng J, Yang L, Yang F et al (2008) Characterization of ST-4821 complex, a unique Neisseria meningitidis clone. Genomics 91(1):78–87
Parkhill J, Achtman M, James KD et al (2000) Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404(6777):502–506
Power PM, Roddam LF, Rutter K et al (2003) Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis. Mol Microbiol 49(3):833–847
Srikhanta YN, Dowideit SJ, Edwards JL et al (2009) Phasevarions mediate random switching of gene expression in pathogenic Neisseria. PLoS Pathog 5(4):e1000400
Tauseef I, Ali YM, Bayliss CD (2013) Phase variation of PorA, a major outer membrane protein, mediates escape of bactericidal antibodies by Neisseria meningitidis. Infect Immun 81(4):1374–1380
Anderson MT, Seifert HS (2013) Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene. PLoS One 8(8):e72183
Seib KL, Jen FE, Tan A et al (2015) Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N(6)-adenine DNA methyltransferases of Neisseria meningitidis. Nucleic Acids Res 43(8):4150–4162
Hood DW, Deadman ME, Jennings MP et al (1996) DNA repeats identify novel virulence genes in Haemophilus influenzae. Proc Natl Acad Sci U S A 93(20):11121–11125
Saunders NJ, Peden JF, Hood DW et al (1998) Simple sequence repeats in the Helicobacter pylori genome. Mol Microbiol 27(6):1091–1098
Jordan PW, Snyder LA, Saunders NJ (2005) Strain-specific differences in Neisseria gonorrhoeae associated with the phase variable gene repertoire. BMC Microbiol 5:21
Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27(2):573–580
Martin P, van de Ven T, Mouchel N et al (2003) Experimentally revised repertoire of putative contingency loci in Neisseria meningitidis strain MC58: evidence for a novel mechanism of phase variation. Mol Microbiol 50(1):245–257
Jen FE, Warren MJ, Schulz BL et al (2013) Dual pili post-translational modifications synergize to mediate meningococcal adherence to platelet activating factor receptor on human airway cells. PLoS Pathog 9(5):e1003377
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Jen, F.EC., Seib, K.L., Tan, A. (2019). Screening DNA Repeat Tracts of Phase Variable Genes by Fragment Analysis. In: Seib, K., Peak, I. (eds) Neisseria meningitidis. Methods in Molecular Biology, vol 1969. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9202-7_6
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DOI: https://doi.org/10.1007/978-1-4939-9202-7_6
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