Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis
Main Article Content
Keywords
pulsed-field gel electrophoresis, deep-well 48-well plate, chromosomal DNA preparation, DNA plugs, karyotyping, genomic fingerprinting
Abstract
Genome analysis using pulsed-field gel electrophoresis (PFGE) has been used in applications ranging from typing bacterial strains to radiobiology to cancer research. While methods for running PFGE have been significantly improved since its invention, the method for preparing chromosomal DNA itself has remained essentially unchanged. This limits the applicability of PFGE, especially when analyses require many samples. We have streamlined sample preparation for routine applications of PFGE through the use of deep-well 48-well plates. Besides saving time, our protocol has the added advantage of reducing the volume of expensive reagents. Our improved protocol enables us to reduce throughput time and simplify the procedure, facilitating wider application of PFGE-based analyses in the laboratory.
Downloads
Metrics
References
2. Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984;37: 67–75.
3. Goering R V. Pulsed field gel electrophoresis: a review of application and interpretation in the molecular epidemiology of infectious disease. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2010;10: 866–875. doi:10.1016/j.meegid.2010.07.023
4. Maurer JJ, Martin G, Hernandez S, Cheng Y, Gerner-Smidt P, Hise KB, et al. Diversity and Persistence of Salmonella enterica Strains in Rural Landscapes in the Southeastern United States. PLoS One. 2015;10: e0128937. doi:10.1371/journal.pone.0128937
5. Janson M, Nordenskjöld M. A constitutional mutation within the retinoblastoma gene detected by PFGE. Clin Genet. 1994;45: 5–10. Available: http://www.ncbi.nlm.nih.gov/pubmed/8149654
6. Zwicker F, Ebert M, Huber PE, Debus J, Weber K-J. A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells. Radiat Oncol. 2011;6: 15. doi:10.1186/1748-717X-6-15
7. Gustafsson A-S, Hartman T, Stenerlöw B. Formation and repair of clustered damaged DNA sites in high LET irradiated cells. Int J Radiat Biol. 2015;91: 820–6. doi:10.3109/09553002.2015.1068463
8. Duck WM, Steward CD, Banerjee SN, McGowan JE, Tenover FC. Optimization of computer software settings improves accuracy of pulsed-field gel electrophoresis macrorestriction fragment pattern analysis. J Clin Microbiol. 2003;41: 3035–3042.
9. Yokoyama E, Uchimura M. Optimal settings of fingerprint-type analysing computer software for the analysis of enterohaemorrhagic Escherichia coli pulsed-field gel electrophoresis patterns. Epidemiol Infect. 2006;134: 1004–1014. doi:10.1017/S0950268806006145
10. PULSENET (Centers for Disease Control and Prevention). Standard Operating Procedure for PulseNet PFGE of Escherichia coli O157:H7, Escherichia coli non - O157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexneri [Internet]. Available: https://www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-pfge-protocol-508c.pdf
11. Kjeldsen MK, Torpdahl M, Pedersen K, Nielsen EM. Development and comparison of a generic multiple-locus variable-number tandem repeat analysis with pulsed-field gel electrophoresis for typing of Salmonella enterica subsp. enterica. J Appl Microbiol. 2015;119: 1707–17. doi:10.1111/jam.12965
12. Feltrin F, Alba P, Kraushaar B, Ianzano A, Argudín MA, Di Matteo P, et al. A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy. Dozois CM, editor. Appl Environ Microbiol. 2016;82: 816–821. doi:10.1128/AEM.02854-15
13. Warren AE, Boulianne-Larsen CM, Chandler CB, Chiotti K, Kroll E, Miller SR, et al. Genotypic and phenotypic variation in Pseudomonas aeruginosa reveals signatures of secondary infection and mutator activity in certain cystic fibrosis patients with chronic lung infections. Infect Immun. 2011;79: 4802–18. doi:10.1128/IAI.05282-11
14. Bidenne C, Blondin B, Dequin S, Vezinhet F. Analysis of the chromosomal DNA polymorphism of wine strains of Saccharomyces cerevisiae. Curr Genet. 1992;22: 1–7.
15. van Dijken J., Bauer J, Brambilla L, Duboc P, Francois J., Gancedo C, et al. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol. 2000;26: 706–714. doi:10.1016/S0141-0229(00)00162-9
Article Sidebar
Article Details
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
Linda Hicks, University of Montana
Division of Biology
Research Associate Professor
