1887

Simple Protocol for Molecular Fingerprinting of Human Oral Microbiota Samples in Lab Classes

    Authors: Ana C. Henriques1, Paolo De Marco1,*
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    Affiliations: 1: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde (IINFACTS), 4585-116 Gandra PRD, Portugal
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Received 02 February 2017 Accepted 27 October 2017 Published 26 January 2018
    • ©2018 Author(s). Published by the American Society for Microbiology
    • [open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.

    • *Corresponding author. Mailing address: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde (IINFACTS), Rua Central de Gandra, 1317, 4585-116, Gandra PRD, Portugal
    Source: J. Microbiol. Biol. Educ. January 2018 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1305
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    Abstract:

    DNA fingerprinting is a major tool in identifying individuals and in evidence matching. However, this technique can be difficult to reproduce in practical classes. Here, we report on distinct PCR profiles obtained when amplifying saliva DNA of a score of distinct individuals with Random Amplified Polymorphic DNA (RAPD)-PCR primer BOXA1R. The RAPD-PCR method is simple and efficient for discrimination between bacterial strains and is used in this instance to obtain personalized fingerprints of each individual’s oral microbiota. We present real results with undergraduate students confirming that this procedure is easily feasible in practical classes. Based on the results presented, we suggest a laboratory activity for undergraduate Molecular Biology or Microbiology students.

References & Citations

1. Versalovic J, Koeuth T, Lupski JR1991Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomesNucleic Acids Res196823683110.1093/nar/19.24.68231762913329316 http://dx.doi.org/10.1093/nar/19.24.6823
2. Shangkuan YH, Yang JF, Lin HC, Shaio MF2000Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strainsJ Appl Microbiol8945246210.1046/j.1365-2672.2000.01134.x11021577 http://dx.doi.org/10.1046/j.1365-2672.2000.01134.x
3. Tacão M, Alves A, Saavedra MJ, Correia A2005BOX-PCR is an adequate tool for typing Aeromonas sppAntonie van Leeuwenhoek8817317910.1007/s10482-005-3450-916096694 http://dx.doi.org/10.1007/s10482-005-3450-9
4. Wang G, Whittam TS, Berg CM, Berg DE1993RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strainsNucleic Acids Res215930593310.1093/nar/21.25.59308290354310476 http://dx.doi.org/10.1093/nar/21.25.5930
5. Rajasundari K, Ilamurugu K, Logeshwaran P2009Genetic diversity in rhizobial isolates determined by RAPDsAfrican J Biotechnol826772681
6. Shangkuan YH, Lin HC1998Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella speciesJ Appl Microbiol8569370210.1111/j.1365-2672.1998.00582.x9812381 http://dx.doi.org/10.1111/j.1365-2672.1998.00582.x
7. Saunders GC, Dukes J, Parkes HC, Cornett JH2001Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analysesClin Chem47475511148176
8. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE1992DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprintingNucleic Acids Res205137514210.1093/nar/20.19.51371408828334296 http://dx.doi.org/10.1093/nar/20.19.5137
9. Welsh J, McClelland M1990Fingerprint genomes using PCR with arbitrary primersNucleic Acids Res186531653510.1093/nar/18.24.7213 http://dx.doi.org/10.1093/nar/18.24.7213
10. Ménard C, Brousseau R, Mouton C1992Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalisFEMS Microbiol Lett9516316810.1111/j.1574-6968.1992.tb05360.x http://dx.doi.org/10.1111/j.1574-6968.1992.tb05360.x
11. Butler JM2005Forensic DNA TypingSecond edElsevier Academic PressBurlington, MA
12. McNamara-Schroeder K, Olonan C, Chu S, Montoya MC, Alviri M, Ginty S, Love JJ2006DNA fingerprint analysis of three short tandem repeat (STR) loci for biochemistry and forensic science laboratory coursesBiochem Mol Biol Educ3437838310.1002/bmb.2006.49403405266521638722 http://dx.doi.org/10.1002/bmb.2006.494034052665
13. Baransel A, Dulger HE, Tokdemir M2004DNA amplification fingerprinting using 10 × polymerase chain reaction buffer with ammonium sulfate for human identificationSaudi Med J2574174515195203
14. Ursell LK, Clemente JC, Rideout JR, Gevers D, Caporaso GJ, Knight R2012The interpersonal and intrapersonal diversity of human associated microbiota in key body sitesJ Allergy Clin Immunol1291204120810.1016/j.jaci.2012.03.010225413613342686 http://dx.doi.org/10.1016/j.jaci.2012.03.010
15. Dawes C2003Estimates, from salivary analyses, of the turnover time of the oral mucosal epithelium in humans and the number of bacteria in an edentulous mouthArch Oral Biol4832933610.1016/S0003-9969(03)00014-112711376 http://dx.doi.org/10.1016/S0003-9969(03)00014-1
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2018-01-26
2018-09-20

Abstract:

DNA fingerprinting is a major tool in identifying individuals and in evidence matching. However, this technique can be difficult to reproduce in practical classes. Here, we report on distinct PCR profiles obtained when amplifying saliva DNA of a score of distinct individuals with Random Amplified Polymorphic DNA (RAPD)-PCR primer BOXA1R. The RAPD-PCR method is simple and efficient for discrimination between bacterial strains and is used in this instance to obtain personalized fingerprints of each individual’s oral microbiota. We present real results with undergraduate students confirming that this procedure is easily feasible in practical classes. Based on the results presented, we suggest a laboratory activity for undergraduate Molecular Biology or Microbiology students.

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Figures

Image of FIGURE 1

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FIGURE 1

Results obtained by five groups of students (A to E). A) 1, 2, 3, 4 = individual saliva DNA samples. 5 = water negative control. B) 1, 3, 4, 5, 6 = individual saliva DNA samples. 7 = water negative control. 2 = same sample as nº 1, loaded twice by mistake. C) 1, 2, 3, 4, 5 = individual saliva DNA samples. 6 = water negative control. D) 1, 2, 3, 4, 5 = individual saliva DNA samples. 6 = water negative control. E) 1, 2, 3, 4 = individual saliva DNA samples. 5 = water negative control. M = molecular weight marker.

Source: J. Microbiol. Biol. Educ. January 2018 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1305
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Image of FIGURE 2

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FIGURE 2

Histogram of grades (0 to 20) from 67 students. 0 to 7 (red) = not proficient; 8 to 15 (blue) = proficient; 16 to 20 (green) = highly proficient. Questions and students’ answer examples can be found in Appendix 6 , with our suggestion for a scoring rubric linked to student learning objectives. Authorization for using students’ grades was obtained from our internal Ethics Committee.

Source: J. Microbiol. Biol. Educ. January 2018 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1305
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