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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.
    • † Supplemental materials available at http://asmscience.org/jmbe
    • *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 JR 1991 Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes Nucleic Acids Res 19 6823 6831 10.1093/nar/19.24.6823 1762913 329316 http://dx.doi.org/10.1093/nar/19.24.6823
2. Shangkuan YH, Yang JF, Lin HC, Shaio MF 2000 Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains J Appl Microbiol 89 452 462 10.1046/j.1365-2672.2000.01134.x 11021577 http://dx.doi.org/10.1046/j.1365-2672.2000.01134.x
3. Tacão M, Alves A, Saavedra MJ, Correia A 2005 BOX-PCR is an adequate tool for typing Aeromonas spp Antonie van Leeuwenhoek 88 173 179 10.1007/s10482-005-3450-9 16096694 http://dx.doi.org/10.1007/s10482-005-3450-9
4. Wang G, Whittam TS, Berg CM, Berg DE 1993 RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains Nucleic Acids Res 21 5930 5933 10.1093/nar/21.25.5930 8290354 310476 http://dx.doi.org/10.1093/nar/21.25.5930
5. Rajasundari K, Ilamurugu K, Logeshwaran P 2009 Genetic diversity in rhizobial isolates determined by RAPDs African J Biotechnol 8 2677 2681
6. Shangkuan YH, Lin HC 1998 Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species J Appl Microbiol 85 693 702 10.1111/j.1365-2672.1998.00582.x 9812381 http://dx.doi.org/10.1111/j.1365-2672.1998.00582.x
7. Saunders GC, Dukes J, Parkes HC, Cornett JH 2001 Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analyses Clin Chem 47 47 55 11148176
8. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE 1992 DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting Nucleic Acids Res 20 5137 5142 10.1093/nar/20.19.5137 1408828 334296 http://dx.doi.org/10.1093/nar/20.19.5137
9. Welsh J, McClelland M 1990 Fingerprint genomes using PCR with arbitrary primers Nucleic Acids Res 18 6531 6535 10.1093/nar/18.24.7213 http://dx.doi.org/10.1093/nar/18.24.7213
10. Ménard C, Brousseau R, Mouton C 1992 Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis FEMS Microbiol Lett 95 163 168 10.1111/j.1574-6968.1992.tb05360.x http://dx.doi.org/10.1111/j.1574-6968.1992.tb05360.x
11. Butler JM 2005 Forensic DNA Typing Second ed Elsevier Academic Press Burlington, MA
12. McNamara-Schroeder K, Olonan C, Chu S, Montoya MC, Alviri M, Ginty S, Love JJ 2006 DNA fingerprint analysis of three short tandem repeat (STR) loci for biochemistry and forensic science laboratory courses Biochem Mol Biol Educ 34 378 383 10.1002/bmb.2006.494034052665 21638722 http://dx.doi.org/10.1002/bmb.2006.494034052665
13. Baransel A, Dulger HE, Tokdemir M 2004 DNA amplification fingerprinting using 10 × polymerase chain reaction buffer with ammonium sulfate for human identification Saudi Med J 25 741 745 15195203
14. Ursell LK, Clemente JC, Rideout JR, Gevers D, Caporaso GJ, Knight R 2012 The interpersonal and intrapersonal diversity of human associated microbiota in key body sites J Allergy Clin Immunol 129 1204 1208 10.1016/j.jaci.2012.03.010 22541361 3342686 http://dx.doi.org/10.1016/j.jaci.2012.03.010
15. Dawes C 2003 Estimates, from salivary analyses, of the turnover time of the oral mucosal epithelium in humans and the number of bacteria in an edentulous mouth Arch Oral Biol 48 329 336 10.1016/S0003-9969(03)00014-1 12711376 http://dx.doi.org/10.1016/S0003-9969(03)00014-1
16. Kort R, Caspers M, Van De Graaf A, Van Egmond W, Keijser B, Roeselers G 2014 Shaping the oral microbiota through intimate kissing Microbiome 2 1 8 10.1186/2049-2618-2-41 http://dx.doi.org/10.1186/2049-2618-2-41
17. Song SJ, Lauber C, Costello EK, Lozupone CA, Humphrey G, Berg-lyons D, Caporaso JG, Knights D, Clemente JC, Nakielny S, Gordon JI, Fierer N, Knight R 2013 Cohabiting family members share microbiota with one another and with their dogs Elife 2 e00458:1 22 10.7554/eLife.00458 http://dx.doi.org/10.7554/eLife.00458
18. Siqueira JF, Fouad AF, Rôças IN 2012 Pyrosequencing as a tool for better understanding of human microbiomes J Oral Microbiol 4 1 15 10.3402/jom.v4i0.10743 http://dx.doi.org/10.3402/jom.v4i0.10743
19. Hasan NA, Young BA, Minard-Smith AT, Saeed K, Li H, Heizer EM, McMillan NJ, Isom R, Abdullah AS, Bornman DM, Faith SA, Choi SY, Dickens ML, Cebula TA, Colwell RR 2014 Microbial community profiling of human saliva using shotgun metagenomic sequencing PLoS One 9 5 e97699 10.1371/journal.pone.0097699 24846174 4028220 http://dx.doi.org/10.1371/journal.pone.0097699
20. Versalovic J, Schneider M, de Bruijn FJ, Lupski JR 1994 Genomic fingerprint of bacteria using repetitive sequence-based polymerase chain reaction Methods Mol Cell Biol 5 25 40

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2018-01-26
2021-01-25

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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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.
  • † Supplemental materials available at http://asmscience.org/jmbe
  • *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

Figures

Simple Protocol for Molecular Fingerprinting of Human Oral Microbiota Samples in Lab Classes
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Citation: Henriques A, De Marco P. 2018. Simple protocol for molecular fingerprinting of human oral microbiota samples in lab classes . J. Microbiol. Biol. Educ. 19(1): doi:10.1128/jmbe.v19i1.1305
/content/jmbe/10.1128/jmbe.v19i1.1305.f1-jmbe-19-1
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.

jmbe/19/1/jmbe-19-1f1_thmb.gif
jmbe/19/1/jmbe-19-1f1.gif
Image of FIGURE 1

Click to view

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
Download as Powerpoint
/content/jmbe/10.1128/jmbe.v19i1.1305.f2-jmbe-19-1
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.

jmbe/19/1/jmbe-19-1f2_thmb.gif
jmbe/19/1/jmbe-19-1f2.gif
Image of FIGURE 2

Click to view

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
Download as Powerpoint

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