The authors would like to thank Kevin Graham for his voice-over and all participants who donated samples for this work.

NOTE: This protocol is only to be used if students or respective legal guardians agreed to the conductance of this protocol as genotypic profiles give insights into genetic relationships. DNA fingerprinting is a common molecular biology method mainly applied to population studies and forensic matters. Therefore, attention should be devoted to keep contamination risks as low as possible. To avoid contamination of the sample with DNA from an outside source or DNases, gloves should be worn, instruments should be thoroughly cleaned or sterilized and solutions should be filter-sterilized or autoclaved before usage.
1. Harvesting of buccal mucosa epithelial cells
CAUTION: Work with saliva and epithelial cells can lead to a transmission of infectious diseases. Therefore, standard and transmission-based precautions should be applied (e.g., the use of appropriate personal protective equipment).
NOTE: Include a positive control, which is provided by the instructor (e.g., DNA extracted by the instructor) and included in the downstream processing steps.
<ol>
	<li>Wait at least 1 h after eating or brushing teeth before specimen collection.</li>
	<li>Label an empty and sterile 2.0 mL microcentrifuge tube.</li>
	<li>Remove one sterile buccal swab from the packaging and rub vigorously on the inside of the cheek for 30-40 times or 30-40 seconds to harvest buccal mucosa epithelial cells.</li>
	<li>Place the tip of the collection swab into the previously labeled sterile microcentrifuge tube and break off the length of plastic that extends beyond the edge, either by hand or using sterile scissors.</li>
	<li>Place the cap securely onto the tube, sealing the collection swab inside.</li>
</ol>
2. Extraction of genomic DNA from human cells
<ol>
	<li>Before extraction, set a thermal mixer or a heating block to 65 &#176;C for sample lysis. 
	CAUTION: Some chemicals used are classified hazardous. Read the safety data sheet carefully and take appropriate safety measures before handling.
	<ol>
		<li>Prepare and sterilize by filtering or autoclaving all required buffers and solutions (lysis solution, 8 M potassium acetate, 2-propanol, 70% ethanol and elution buffer). 
		NOTE: All centrifugation steps should be performed at room temperature (20-30 &#176;C) unless specified.</li>
	</ol>
	</li>
	<li>Add 500 &#956;L of lysis solution (50 mM Tris/HCl, pH 8.0; 10 mM ethylenediamine tetra-acetic acid (EDTA); 2% sodium dodecyl sulfate (SDS)) to the buccal swab, making sure that the sample is completely immersed in the lysis solution.
	<ol>
		<li>Vortex vigorously for at least 5 s.</li>
		<li>Incubate samples at 65 &#176;C in a thermal mixer for 10 min.</li>
		<li>Mix sample 3-4 times by pulse-vortexing for 5 s during incubation.</li>
		<li>Remove swab from the lysis buffer, press the swab against the inside of the tube to obtain maximum sample volume.</li>
	</ol>
	</li>
	<li>Add 100 &#956;L of 8 M potassium acetate to lysed cells.
	<ol>
		<li>Mix thoroughly by inverting the tube until there is a white precipitate.</li>
		<li>Incubate for 5 min at room temperature.</li>
	</ol>
	</li>
	<li>Centrifuge the sample for 5 min at 18,000 x g.</li>
	<li>Transfer 450 &#956;L of the supernatant into a clean and sterile 1.5 mL microcentrifuge tube.</li>
	<li>Add 450 &#956;L of 2-propanol and mix thoroughly by inverting the tube (precipitation of the DNA).</li>
	<li>Incubate for 5 min at room temperature.</li>
	<li>Centrifuge for 5 min at 18,000 x g.</li>
	<li>Discard the supernatant and invert the tube on a clean paper towel to dry the pellet and to avoid cross-contamination.</li>
	<li>Incubate the DNA for 5 min at 65 &#176;C in a heating block to dry the pellet completely.</li>
	<li>To wash the DNA, add 500 &#956;L of 70% ethanol.</li>
	<li>Centrifuge for 1 min at 18,000 x g.</li>
	<li>Discard the supernatant and invert the tube on a clean paper towel to dry the pellet.</li>
	<li>Add 30 &#956;L of resuspension buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA) to the pellet.</li>
	<li>Incubate the DNA for 10 min at 65 &#176;C in a heating block to inactivate DNases.</li>
</ol>
3. Amplifying the D1S80 VNTR locus using PCR
NOTE: Record the number of samples that will be used and prepare a worksheet with the required reagents and their volumes before collecting the necessary plastics and other materials. Label the sterile tubes/strips/plates to be used for the PCR with sample numbers. Remember to include a negative control using H2O instead of DNA and a positive control (e.g.,&#160;using the DNA provided by the instructor) to validate the PCR.
<ol>
	<li>Prepare the 1x PCR master mix containing 10 &#956;L of 5x PCR reaction buffer (containing 15 mM MgCl2), 1 &#956;L of deoxyribonucleotide triphosphate (dNTPs) (10 mM), 5 &#956;L (10 pmol) of each primer pMCT118-f and pMCT118-r (forward - 5&#39;-GAAACTGGCCTCCAAACACTGCCCGCCG-3&#39;, reverse - 5&#39;-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3&#39;) according to Kasai et al.2, 23.8 &#956;L of ultrapure H2O, and 0.2 &#956;L of Taq DNA polymerase (5 U/&#181;L). 
	NOTE: The primers pMCT118-f/pMCT118-r were designed on the flanking regions of the D1S80 VNTR region amplifying the whole locus.</li>
	<li>Label the PCR tubes/strips/plate with the sample numbers to be used.</li>
	<li>Aliquot 45 &#956;L of the master mix to each labeled PCR sample tube or well.</li>
	<li>Add 5 &#956;L of the DNA template to the master mix in each PCR tube/plate well to acquire a total volume of 50 &#181;L. Change the pipette tip for every DNA sample to avoid cross contamination.</li>
	<li>Include a no template control (NTC) by using ultrapure H2O instead of DNA.</li>
	<li>Close PCR tubes/strips or seal plate and mix.</li>
	<li>Centrifuge the PCR tubes/strips/plates for 20 s using a tabletop centrifuge. 
	NOTE: PCR conditions given in this protocol were optimized using the DNA polymerase and the PCR thermal cycler used. In general, PCR conditions must be adapted to the DNA polymerase. The standard extension time for a Taq DNA polymerase is 1 min/kb.</li>
	<li>Place the sample tubes/strips/plate in the thermocycler and incubate reactions using the following conditions (DNA polymerase extension time): 1 cycle of 95 &#176;C for 2 min; 25 cycles of 94 &#176;C for 15 s, 60 &#176;C for 15 s, 72 &#176;C for 30 s; 1 cycle of 72 &#176;C for 10 min.</li>
	<li>When the program is complete, remove the products from the thermocycler and store at 4 &#176;C overnight or -20 &#176;C until electrophoresis.</li>
</ol>
4. Agarose gel electrophoresis of PCR products
<ol>
	<li>Prepare a 1.5% agarose gel.
	<ol>
		<li>Use 1.5 g of agarose powder and mix it with 100 mL of 1x Tris-acetate-EDTA (TAE) buffer solution in a flask.</li>
		<li>Heat the mixture for about 1.5-2 min in a microwave oven (600 W). Swirl the contents and heat again, if necessary, to completely dissolve the agarose. Cool slightly and add 2 &#956;L of PeqGreen to the agarose.</li>
		<li>Pour the gel in a form using a comb with enough wells for all samples and add at least one molecular weight marker. 
		CAUTION: Boiling retardation may occur. Shake the flask carefully when resuspending the agarose which has not been dissolved yet. 
		NOTE: PeqGreen is a non-toxic dye for the detection of nucleic acids. It is applicable for staining of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as well as RNA. The sensitivity is comparable to ethidium bromide.</li>
	</ol>
	</li>
	<li>Remove the PCR products from 4 &#176;C and centrifuge for about 10 s.</li>
	<li>Load the wells of the gel with 10 &#956;L sample. Do not overload the gel. 
	NOTE: The DNA polymerase buffer also includes compounds that increases sample density so that samples can be loaded directly onto gels without the need for a loading dye. This allows the sample to sink into the well and dyes help to track how far the DNA sample has migrated.
	<ol>
		<li>Add a molecular weight standard to the flanking wells (preferably a 50 bp molecular weight standard). 
		NOTE: Store the residual PCR samples at -20 &#176;C in case the agarose gel electrophoresis has to be repeated.</li>
	</ol>
	</li>
	<li>Run the gel in 1x TAE running buffer at 150 V (constant) for approximately 40 min or until the lower yellow dye front that travels at around 50 bp reaches the lower end of the gel.</li>
	<li>Image the gel while blue light or ultraviolet (UV) light is applied and record an image for fragment length analysis. 
	CAUTION: UV light can damage your eyes and skin. Always wear protective clothing and UV safety glasses when using a UV light box.</li>
	<li>Dispose the gel in accordance with the institutional hazardous materials policy.</li>
</ol>
5. Analysis of fragment length results
NOTE: Use linear regression analysis to estimate the lengths of the fragments.
<ol>
	<li>For sizing the D1S80 PCR fragments, place a ruler on the gel photograph over the 50 bp molecular weight standard lane such that the top of the ruler lines up with the bottom of the well into which the sample was loaded (Figure 1). 
	NOTE: To calculate the linear regression equation for the molecular weight standard (50 bp molecular weight standard) a spreadsheet program was used.</li>
	<li>Record the distance from the well (e.g., in cm) for each band of the 50 bp molecular weight standard in a table using a spreadsheet program (Figure 2).</li>
	<li>Determine the log (e.g., base 10) of each fragment size of the 50 bp molecular weight standard and enter the log values into the table (Figure 2).</li>
	<li>Plot each data point to a graph with the log of the band sizes on the vertical axis (y-axis) and the measured run distance from the top of the gel to each band of the molecular weight standard on the horizontal axis (x-axis) using a scatterplot (Figure 3).</li>
	<li>Fit a trendline (linear regression line) and show the regression equation (y = ax + b) and the R2 value on the graph (Figure 4).</li>
	<li>Measure the distance migrated (e.g., in cm) for each D1S80 amplicon&#160;(Figure 5).</li>
	<li>Estimate the size of each D1S80 amplicon&#160;using the regression equation: y = ax + b 
	where y = the log of the fragment size 
	&#160; &#160; &#160; &#160; &#160; &#160;a = the slope of the line (calculated in point 5) 
	&#160; &#160; &#160; &#160; &#160; &#160;x = the distance from the well (in cm) 
	&#160; &#160; &#160; &#160; &#160; &#160;b = the point where the regression line intercepts the y-axis (calculated in point 5) 
	&#8203;NOTE: Since the y-value represents the log of the fragment size, the antilog (10y) must be calculated to obtain the fragment size in bp of the D1S80 amplicons.</li>
</ol>
6. Estimation of the number of repeat units in the alleles of the tested individuals
NOTE: The D1S80 repeat unit is 16 bp in length. The smallest known allele for D1S80 has 14 repeats. The amplicon scheme described here produces amplicons with flanking regions adding up an extra 145 bp to the final size.
<ol>
	<li>Use Table 1 to estimate how many repeat units are contained in each PCR fragment. The size extrapolated using the linear regression should be within 8 bp of any particular allele.</li>
	<li>Record the genotype of each tested individual as a combination of allele repeat size numbers.</li>
</ol>

<ol>
	<li>Jeffreys, A. J., Wilson, V., Thein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypervariable+'minisatellite'+regions+in+human+DNA.">Hypervariable 'minisatellite' regions in human DNA.</a> Nature. 314 (6006), 67-73 (1985).</li><li>Kasai, K., Nakamura, Y., White, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+a+variable+number+of+tandem+repeats+(VNTR)+locus+(pMCT118)+by+the+polymerase+chain+reaction+(PCR)+and+its+application+to+forensic+science.">Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science.</a> Journal of Forensic Science. 35 (5), 1196-1200 (1990).</li><li>Balamurugan, K., Tracey, M. L., Heine, U., Maha, G. C., Duncan, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+at+the+human+D1S80+minisatellite+locus.">Mutation at the human D1S80 minisatellite locus.</a> The Scientific World Journal. 2012 (917235), (2012).</li><li>Herrera, R. J., Adrien, L. R., Ruiz, L. M., Sanabria, N. Y., Duncan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1S80+single-locus+discrimination+among+African+populations.">D1S80 single-locus discrimination among African populations.</a> Human Biology. 76 (1), 87-108 (2004).</li><li>Lauritzen, S. L., Mazumder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Informativeness+of+genetic+markers+for+forensic+inference+-+An+information+theoretic+approach.">Informativeness of genetic markers for forensic inference - An information theoretic approach.</a> Forensic Science International: Genetics Supplement Series. 1 (1), 652-653 (2008).</li><li>Budowle, B., Chakraborty, R., Giusti, A. M., Eisenberg, A. J., Allen, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+VNTR+locus+D1S80+by+the+PCR+followed+by+high-resolution+PAGE.">Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE.</a> American Journal of Human Genetics. 48 (1), 137 (1991).</li><li>Budowle, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1S80+population+data+in+African+Americans,+Caucasians,+southeastern+Hispanics,+southwestern+Hispanics,+and+Orientals.">D1S80 population data in African Americans, Caucasians, southeastern Hispanics, southwestern Hispanics, and Orientals.</a> Journal of Forensic Science. 40 (1), 38-44 (1995).</li><li>Verbenko, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymorphisms+at+locus+D1S80+and+other+hypervariable+regions+in+the+analysis+of+Eastern+European+ethnic+group+relationships.">Polymorphisms at locus D1S80 and other hypervariable regions in the analysis of Eastern European ethnic group relationships.</a> Annals of Human Biology. 33 (5-6), 570-584 (2006).</li><li>Walsh, S. J., Eckhoff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Australian+Aboriginal+population+genetics+at+the+D1S80+VNTR+locus.">Australian Aboriginal population genetics at the D1S80 VNTR locus.</a> Annals of Human Biology. 34 (5), 557-565 (2007).</li><li>Limborska, S. A., Khrunin, A. V., Flegontova, O. V., Tasitz, V. A., Verbenko, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+genetic+diversity+in+D1S80+revealed+by+SNP-VNTR+haplotyping.">Specificity of genetic diversity in D1S80 revealed by SNP-VNTR haplotyping.</a> Annals of Human Biology. 38 (5), 564-569 (2011).</li><li>Sajib, A. A., Yeasmin, S., Akter, M., Uddin, M. A., Akhteruzzaman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+Bangladeshi+population+with+reference+to+D1S80+VNTR+locus.">Phylogenetic analysis of Bangladeshi population with reference to D1S80 VNTR locus.</a> Bioresearch Communications. 2 (1), 146-151 (2016).</li><li>K&#246;seler, A., Atalay, A., Atalay, E. &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele+frequency+of+VNTR+locus+D1S80+observed+in+Denizli+province+of+Turkey.">Allele frequency of VNTR locus D1S80 observed in Denizli province of Turkey.</a> Biochemical genetics. 47 (7-8), 540-546 (2009).</li><li>Mastana, S. S., Papiha, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1S80+distribution+in+world+populations+with+new+data+from+the+UK+and+the+Indian+sub-continent.">D1S80 distribution in world populations with new data from the UK and the Indian sub-continent.</a> Annals of Human Biology. 28 (3), 308-318 (2001).</li><li>Okuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Japanese+propositus+with+D--+phenotype+characterized+by+the+deletion+of+both+the+RHCE+gene+and+D1S80+locus+situated+in+chromosome+1p+and+the+existence+of+a+new+CE-D-CE+hybrid+gene.">A Japanese propositus with D-- phenotype characterized by the deletion of both the RHCE gene and D1S80 locus situated in chromosome 1p and the existence of a new CE-D-CE hybrid gene.</a> Journal of Human Genetics. 45 (3), 142-153 (2000).</li><li>Campbell, A. M., Williamson, J. H., Padula, D., Sundby, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+PCR+&amp;+a+Single+Hair+to+Produce+a+"DNA+Fingerprint.".">Use PCR &amp; a Single Hair to Produce a "DNA Fingerprint.".</a> The American Biology Teacher. 59 (3), 172-178 (1997).</li><li>Jackson, D. D., Abbey, C. S., Nugent, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+profiling+of+the+D1S80+locus:+A+forensic+analysis+for+the+undergraduate+biochemistry+laboratory.">DNA profiling of the D1S80 locus: A forensic analysis for the undergraduate biochemistry laboratory.</a> Journal of Chemical Education. 83 (5), 774 (2006).</li><li>Mansoor, S. K., Hussein, E. F., Ibraheem, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+the+Variable+Number+of+Tandem+Repeats+(VNTR)+in+DNA+fingerprinting+and+its+application+biological+sciences.">Use the Variable Number of Tandem Repeats (VNTR) in DNA fingerprinting and its application biological sciences.</a> EurAsian Journal of BioSciences. 14 (2), 2835-2839 (2020).</li><li>Beier, K., Schnorrer, S., Hoppe, N., Lenk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ethical+and+legal+regulation+of+human+tissue+and+biobank+research+in+Europe.">The ethical and legal regulation of human tissue and biobank research in Europe.</a> Proceedings of the Tiss. EU Project. , (2011).</li><li>Aida, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+variations+in+6+mucosal+cell+types+of+gastric+tissue+observed+using+a+novel+quantitative+fluorescence+in+situ+hybridization+method.">Telomere length variations in 6 mucosal cell types of gastric tissue observed using a novel quantitative fluorescence in situ hybridization method.</a> Human Pathology. 38 (8), 1192-1200 (2007).</li><li>Theda, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+the+cellular+content+of+saliva+and+buccal+swab+samples.">Quantitation of the cellular content of saliva and buccal swab samples.</a> Scientific Reports. 8 (1), 1-8 (2018).</li><li>McMichael, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+from+buccal+swabs+suitable+for+high-throughput+SNP+multiplex+analysis.">DNA from buccal swabs suitable for high-throughput SNP multiplex analysis.</a> Journal of biomolecular techniques: JBT. 20 (5), 232 (2009).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>Destro-Bisol, G., Capelli, C., Belledi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+microevolutionary+patterns+from+allele-size+frequency+distributions+of+minisatellite+loci:+a+worldwide+study+of+the+APOB+3'hypervariable+region+polymorphism.">Inferring microevolutionary patterns from allele-size frequency distributions of minisatellite loci: a worldwide study of the APOB 3'hypervariable region polymorphism.</a> Human Biology. 72 (5), 733-751 (2000).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+alleles+in+the+3'end+region+of+human+apoB+gene.">Structure and function of alleles in the 3'end region of human apoB gene.</a> Chinese Medical Journal. 112 (3), 221-223 (1999).</li><li>Renges, H. -. H., Peacock, K., Dunning, A. M., Talmud, P., Humphries, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+relationship+between+the+3+'-VNTR+and+diallelic+apolipoprotein+B+gene+polymorphisms:+Haplotype+analysis+in+individuals+of+European+and+South+Asian+origin.">Genetic relationship between the 3 '-VNTR and diallelic apolipoprotein B gene polymorphisms: Haplotype analysis in individuals of European and South Asian origin.</a> Annals of Human Genetics. 56 (1), 11-33 (1992).</li><li>Kravchenko, S. A., Maliarchuk, O. S., Livshits, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+genetics+study+of+the+allelic+polymorphism+in+the+hypervariable+region+of+the+apolipoprotein+B+gene+in+the+population+of+different+regions+of+Ukraine.">A population genetics study of the allelic polymorphism in the hypervariable region of the apolipoprotein B gene in the population of different regions of Ukraine.</a> Tsitologiia i Genetika. 30 (5), 35-41 (1996).</li></ol>

The authors have no conflict of interest to disclose.

Here we described a simple and cost-efficient method for implementing molecular fingerprinting in undergraduate practical classes.
To screen for genetic variation at the D1S80 locus, human biological sample collection along with DNA extraction and analysis is required. It is essential that the ethical use of human biological specimens is ensured throughout the whole process. Sample management is controlled within a comprehensive regulatory framework which ensures the correct use of samples and associated data18 (e.g., consent for the use of human biological material). Participants must be properly informed about the use of their samples, the risk of discovery of anomalies in genetic relations (e.g., for related individuals), privacy protection and intentions for future storage of the biological specimens and data. All donors (students or colleagues) must give consent freely and understand the right to withdraw without giving reason. In general, it is indispensable to make oneself familiar with the respective guidelines and regulations for human sample management before conducting this lab class.
For the collection of buccal mucosa epithelial cells in undergraduate laboratory courses, care must be taken to avoid contamination of the DNA samples with DNA from the operator or with DNases. It is recommended that lab coats, gloves and protective glasses are always used as well as sterile swabs and microcentrifuge tubes. Buccal swab-based cell harvesting is considered a convenient and cost-effective method for the collection of genetic material suitable for PCR-based VNTR analysis, as it is relatively inexpensive and noninvasive. Beside buccal swabs, saliva is the most common oral sampling method for medical research. It has been shown that buccal swabs contain a higher proportion of epithelial cells than saliva, making them more reliable in providing sufficient quantity and quality of DNA for PCR-based D1S80 VNTR analysis19,20. Additionally, buccal swabs can be mailed out after self-collection overcoming geographical impediments when used, for example, in population diversity studies21.
DNA extraction has become relatively easy due to the development of extraction kits from different companies. Nevertheless, the use of those kits may not be suitable in laboratory classes due to limited financial resources. Here, we presented an easy, fast and cost-efficient method for DNA extraction from human samples without the need of a commercially available DNA extraction kit. The DNA extraction method described does not use organic solvents, instead cellular proteins are removed by increasing the salt concentration using an 8 M potassium acetate solution. This method also allows for handling of several samples at once and yields in sufficient DNA for several genotype analyses for each sample.
PCR is a common technique in many molecular laboratories. While generally trouble-free, there are pitfalls that may complicate the reaction producing spurious results, which have been discussed elsewhere22. PCR conditions presented here have appeared very robust in the face of poor template DNA quality, but lack of PCR amplification was nevertheless occasionally observed when using templates with extremely low DNA concentrations due to poor buccal epithelial cell harvesting. The DNA primers used in this study can be ordered from different companies, such as those cited in the table of materials at the end of this paper. Special care must be taken when working with DNA primers in order to avoid contamination with DNases or DNA from the operator. PCR products should also be kept cold when not in use.
Another critical step is the agarose gel electrophoresis. Fragments differing by only one repeat unit of 16 bp may not be separated in the gel electrophoresis and thus appear as a single band. In this case, a proper determination of the number of repeat units of the D1S80 alleles tested cannot be assured. Therefore, the run time of the gel should be increased while the voltage must be reduced and the gel concentration may be increased to provide a better resolution and separation of the single bands.
It is worth mentioning that not all alleles for an VNTR locus contain complete repeat units. Non-consensus alleles (microvariants) that contain incomplete repeat units are common at most VNTR loci and their sizes fall in between sizes of alleles with full repeat units. These microvariants are barely detectable by agarose gel electrophoresis. In contrast, techniques like polyacrylamide gels or capillary electrophoresis can resolve alleles that differ by one to a few repeat units or microvariants12,23,24,25,26. However, the latter techniques are less suitable for undergraduate laboratory classes as they have many disadvantages including use of hazardous compounds, complex preparation and lack of equipment. For the fragment size determination, special care must be taken when measuring the distance of the migrated DNA fragments. If the bands are too diffuse to be measured precisely, the calculation of the D1S80 allele fragment size by linear regression may be incorrect resulting in the wrong estimation of repeat unit numbers. In that case a re-run of the agarose gel electrophoresis after optimizing the gel conditions is advisable as previously described by Lorenz and co-workers22.
The entire D1S80 VNTR analysis procedure presented here, from buccal epithelial cell harvesting to fragment length evaluation, may be completed in a single working day. This protocol is a robust, cost-efficient and easy-to-use method suitable for undergraduate practical laboratory classes.

Molecular fingerprinting, also referred to as DNA fingerprinting, was introduced by Sir Alec Jeffreys while working in the Department of Genetics at the University of Leicester in 19841. It is based on the 0.1% of the human genome that differs between individuals and is comprised of approximately three million variants. These unique differences in the genotype allow for the differentiation between individuals and can therefore function as a genetic fingerprint except for monozygotic twins. Consequently, DNA fingerprinting is used for the estimation of relatedness of different individuals that is applied for example in paternity testing or in population diversity studies. In our laboratory classes we aimed to convey the concept of DNA fingerprinting and allele frequencies. The method described here demonstrates a reliable and robust method for genetic fingerprinting by analyzing the Variable Number of Tandem Repeats (VNTR) at the D1S80 locus. The method comprises the extraction of DNA from buccal mucosa epithelial cells and subsequent Polymerase Chain Reaction (PCR) to amplify the D1S80 locus, followed by visualization of fragment length differences on an agarose gel.
The D1S80 VNTR minisatellite locus is highly variable and located at the telomeric region of chromosome 1 (1p36-p35). It was identified and described by Karsai and co-workers in 1990 as a locus with a core repeat unit of 16 bp, allowing for the separation of alleles differing by just one unit2. Furthermore, D1S80 shows a high degree of variation with a mutation rate of approximately 7.77 x 10-5&#160;3. D1S80 has a high degree of heterozygosity4,5 (e.g., 80.8% heterozygosity for Caucasians6 and up to 87% heterozygosity for African-Americans7). Additionally, the D1S80 locus is polymorphic in most populations, with typically over 15 different alleles carrying 14 to 41 repeats each. The frequency of D1S80 alleles varies between populations. The allele with 24 repeat units (allele 24) is most frequent in European and Asian populations, whereas allele 21 is most common in African populations4,7,8,9,10. Consequently, the allele frequency distributions are diagnostic for different human populations and need to be taken into account for the estimation of relatedness (e.g., in paternity tests).
PCR based amplification of the D1S80 VNTR locus has been a very useful method in forensic science, paternity tests, disease analysis and population diversity studies11,12,13,14. While in forensics today the use of VNTRs has been replaced by short tandem repeats, the D1S80 VNTR locus is widely used in the determination of origins and genetic relations among and between populations4,8,9,11. Furthermore, it is often used to teach DNA fingerprinting in practical laboratory classes15,16. The method described here represents a robust, cost-effective and easy-to-use method with a very high success rate in undergraduate laboratory classes. The purpose of this article is to provide an overview of the workflow for the molecular analysis of the human D1S80 minisatellite locus from buccal mucosa epithelial cells. It includes demonstration of techniques, simplified protocols and practical suggestions described in previously published works2,17.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Sarstedt</td><td>72,706</td><td>autoclaved</td></tr><tr><td>2 mL microcentrifuge tube</td><td>Sarstedt</td><td>7,26,95,500</td><td>autoclaved</td></tr><tr><td>2-propanol</td><td>Fisher chemical</td><td>PI7508/17</td><td></td></tr><tr><td>5x PCR buffer</td><td>Promega</td><td>M7845</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma/Merck</td><td>A6283-500ML</td><td></td></tr><tr><td>Agarose</td><td>BioBudget</td><td>10-35-1020</td><td></td></tr><tr><td>Buccal swabs</td><td>BioBudget</td><td>57-BS-05-600</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td>5430</td></tr><tr><td>Combs</td><td>BioRad</td><td></td><td></td></tr><tr><td>dNTPs (10 mM)</td><td>Invitrogen</td><td>R0192</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth</td><td>8043.1</td><td></td></tr><tr><td>Ethanol</td><td>Honeywell</td><td>32221-2.5L</td><td></td></tr><tr><td>Gel caster</td><td>BioRad</td><td></td><td></td></tr><tr><td>Gel chamber</td><td>BioRad</td><td></td><td>SubCell GT</td></tr><tr><td>Gel Doc XR+</td><td>BioRad</td><td></td><td></td></tr><tr><td>Geltray</td><td>BioRad</td><td></td><td>SubCell GT</td></tr><tr><td>GeneRuler 50 bp DNA Ladder</td><td>Thermo Fisher</td><td>SM0371</td><td></td></tr><tr><td>Go-Taq Polymerase</td><td>Promega</td><td>M7845</td><td></td></tr><tr><td>Heating block</td><td>Eppendorf</td><td>Thermomixer</td><td>1.5 mL and 2 mL</td></tr><tr><td>Microwave</td><td>Sharp</td><td></td><td>R-941STW</td></tr><tr><td>peqGreen</td><td>VWR</td><td>&amp;nbsp;732-3196&amp;nbsp;</td><td></td></tr><tr><td>Pipettes</td><td>Gilson</td><td></td><td>1000 &amp;micro;L, 200 &amp;micro;L, 20 &amp;micro;L, 2 &amp;micro;L</td></tr><tr><td>Potassium acetate</td><td>Arcos Organics</td><td>217100010</td><td></td></tr><tr><td>PowerPac power supply</td><td>BioRad</td><td></td><td>100-120/220-240V</td></tr><tr><td>Primers</td><td>Sigma/Merck</td><td></td><td></td></tr><tr><td>Scale</td><td>Kern</td><td></td><td>PCB 2.5 kg</td></tr><tr><td>SDS</td><td>Arcos Organics</td><td>218591000</td><td></td></tr><tr><td>Shaker</td><td>IKA labortechnik</td><td></td><td>IKA RH basic</td></tr><tr><td>Tabletop centrifuge</td><td>myFuge</td><td></td><td></td></tr><tr><td>Thermal Cycler</td><td>BioRad</td><td></td><td>C100 Touch</td></tr><tr><td>Tris</td><td>VWR</td><td>103156x</td><td></td></tr><tr><td>Vortex mixer</td><td>LMS</td><td></td><td>VTX-3000L</td></tr></tbody></table>

application of dna fingerprinting using the d1s80 locus in lab classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Using the described protocol, D1S80 VNTR marker analysis was performed on human genomic DNA extracted from buccal mucosa epithelial cells harvested by swab sampling (Figure 6). Following the amplification using PCR, a typical representation of an agarose gel containing the D1S80 amplicons is shown in Figure 1. Lane 1 shows the 50 bp molecular weight standard. Next to the molecular weight standard, PCR products from eight student samples are visualized. Lane 10 shows the NTC in which water was used instead of template DNA. Most of the analyzed samples clearly display two bands, representing individuals heterozygous for the D1S80 locus. Lane 2 and Lane 9 show a single band representing individuals homozygous for the D1S80 locus. Lane 5 is showing an ambiguous single band which is much wider compared to the other bands. This could be a result of two D1S80 alleles differing in only one repeat unit. For further analysis, the sample in lane 5 will be considered as a single band, representing a homozygous individual.
Downstream gel electrophoresis fragment analysis performed on verified PCR products was used for size calling of the D1S80 VNTR locus of different student samples. Interpretation of the amplicon sizes obtained by PCR analysis relies on the molecular weight standard used. The distance from the well for each band of the molecular weight standard was measured (Figure 1) and recorded (Figure 2). Based on the size and migration distance the size of the individual bands can be calculated using a linear regression analysis. The log (base 10) was determined for each band in the student samples (Figure 5) and the respective antilog represents the number of bp for each amplicon. The number of repeat units can be calculated according to the amplicon size obtained (Table 2).
For each student sample the amplicon sizes were calculated by linear regression and each band could be easily assigned to a particular allele as shown in Table 2. The information on the D1S80 allele sizes can then be used to determine allele frequencies amongst the small population subset of undergraduate students. The 28-repeat allele is the most frequent among the students due to two homozygous individuals for this repeat allele (individuals 1 and 4). The second most frequent allele is the 18-repeat allele, which is carried by the homozygous individual 8 and by the heterozygous individual 6. Low frequency alleles are the 21-, 26- and 30-repeat alleles found only once in individuals 6, 5 and 3, respectively. The allele frequency patterns of the D1S80 VNTR locus of the small student population can further be compared to the allele frequency of larger human populations, for example populations originating from different continents4,7,8,9,10.
Among the students, individuals 2 and 3 share the 24-repeat allele, individuals 2 and 7 share the 20-repeat allele and individuals 5 and 7 share the 23-repeat allele. Interestingly, the two D1S80 homozygous individuals (individual 1 and 4) share the 28-repeat allele. Matching alleles between two individuals can indicate a potential relatedness. However, shared alleles can also occur by chance. Thus, it is crucial to determine the likelihood of an allele match between two individuals. The likelihood depends on the allele frequency of individual alleles in the general population and statistical approaches should be employed to attach a statistical weight to VNTR marker analysis such as the Bayesian approach. In summary, the presented fingerprinting method can be utilized in hands-on class practicals to teach the use of VNTRs in DNA fingerprinting and its application in biological science.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62305/62305fig01.jpg" /> 
Figure 1: Representation of an agarose gel after electrophoresis of D1S80 amplification products with a ruler placed beside the 50 bp molecular weight standard lane. Lane 1 contains the 50 bp molecular weight standard, while lanes 2-9 contain PCR reaction products. Lane 10 contains the no template control (NTC). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62305/62305fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62305/62305fig02.jpg" /> 
Figure 2: Determination of DNA fragment migration distance and log of each fragment size of the 50 bp molecular weight standard. The distance from the well (in cm) for each band of the 50 bp molecular weight standard is recorded and the log (base 10) for each fragment is determined. Values are entered into a table using a spreadsheet program. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62305/62305fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62305/62305fig03.jpg" /> 
Figure 3: Scatter plot generated by plotting DNA fragment migration distance of the molecular weight standard on the x-axis against the log (base 10) of the size of each fragment on the y-axis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62305/62305fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62305/62305fig04.jpg" /> 
Figure 4: Exploitation of the linear regression line and regression equation as well as the R2 value for the data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62305/62305fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62305/62305fig05.jpg" /> 
Figure 5: Determination of the size of the D1S80 amplicons. The distance&#160;from the well for each fragment is entered into the regression equation. The antilog of the y-value represents the fragment size in bp. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62305/62305fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62305/62305fig6v2.jpg" /> 
Figure 6: Suggested timeline and workflow for D1S80 VNTR analysis. The entire D1S80 VNTR analysis procedure presented here, from buccal epithelial cell harvesting to fragment length evaluation, may be completed within a single working day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62305/62305fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Allele size (in bp)</td>
			<td>Number of repeats</td>
		</tr>
		<tr>
			<td>369</td>
			<td>14</td>
		</tr>
		<tr>
			<td>385</td>
			<td>15</td>
		</tr>
		<tr>
			<td>401</td>
			<td>16</td>
		</tr>
		<tr>
			<td>417</td>
			<td>17</td>
		</tr>
		<tr>
			<td>433</td>
			<td>18</td>
		</tr>
		<tr>
			<td>449</td>
			<td>19</td>
		</tr>
		<tr>
			<td>465</td>
			<td>20</td>
		</tr>
		<tr>
			<td>481</td>
			<td>21</td>
		</tr>
		<tr>
			<td>497</td>
			<td>22</td>
		</tr>
		<tr>
			<td>513</td>
			<td>23</td>
		</tr>
		<tr>
			<td>529</td>
			<td>24</td>
		</tr>
		<tr>
			<td>545</td>
			<td>25</td>
		</tr>
		<tr>
			<td>561</td>
			<td>26</td>
		</tr>
		<tr>
			<td>577</td>
			<td>27</td>
		</tr>
		<tr>
			<td>593</td>
			<td>28</td>
		</tr>
		<tr>
			<td>609</td>
			<td>29</td>
		</tr>
		<tr>
			<td>625</td>
			<td>30</td>
		</tr>
		<tr>
			<td>641</td>
			<td>31</td>
		</tr>
		<tr>
			<td>657</td>
			<td>32</td>
		</tr>
		<tr>
			<td>673</td>
			<td>33</td>
		</tr>
		<tr>
			<td>689</td>
			<td>34</td>
		</tr>
		<tr>
			<td>705</td>
			<td>35</td>
		</tr>
		<tr>
			<td>721</td>
			<td>36</td>
		</tr>
		<tr>
			<td>737</td>
			<td>37</td>
		</tr>
		<tr>
			<td>753</td>
			<td>38</td>
		</tr>
		<tr>
			<td>769</td>
			<td>39</td>
		</tr>
		<tr>
			<td>785</td>
			<td>40</td>
		</tr>
		<tr>
			<td>801</td>
			<td>41</td>
		</tr>
	</tbody>
</table>
Table 1: Amplicon size for each D1S80 VNTR locus. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Individual</td>
			<td>Allele</td>
			<td>Distance (in cm)</td>
			<td>Size (in bp)</td>
			<td>Number of repeat units</td>
		</tr>
		<tr>
			<td>1</td>
			<td>homozygous</td>
			<td>5</td>
			<td>600</td>
			<td>28</td>
		</tr>
		<tr>
			<td>2</td>
			<td>heterozygous</td>
			<td>5.2 ; 5.5</td>
			<td>535, 458</td>
			<td>24, 20</td>
		</tr>
		<tr>
			<td>3</td>
			<td>heterozygous</td>
			<td>4.9 ; 5.2</td>
			<td>626, 535</td>
			<td>30, 24</td>
		</tr>
		<tr>
			<td>4</td>
			<td>homozygous</td>
			<td>5</td>
			<td>600</td>
			<td>28</td>
		</tr>
		<tr>
			<td>5</td>
			<td>heterozygous</td>
			<td>5.1 ; 5.3</td>
			<td>564, 508</td>
			<td>26, 23</td>
		</tr>
		<tr>
			<td>6</td>
			<td>heterozygous</td>
			<td>5.4 ; 5.6</td>
			<td>483, 435</td>
			<td>21, 18</td>
		</tr>
		<tr>
			<td>7</td>
			<td>heterozygous</td>
			<td>5.3 ; 5.5</td>
			<td>508, 458</td>
			<td>23, 20</td>
		</tr>
		<tr>
			<td>8</td>
			<td>homozygous</td>
			<td>5.6</td>
			<td>435</td>
			<td>18</td>
		</tr>
	</tbody>
</table>
Table 2: Allele composition of the different individuals tested. The number of repeat units can be approximated according to the amplicon size obtained by linear regression analysis.

Watch this Scientific Journal Video about Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes at JoVE.com

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

application-of-dna-fingerprinting-using-the-d1s80-locus-in-lab-classes

Nanyang Technological University

Research

JoVE Journal

Genetics

19.7K Views.  Heinrich-Heine-University. Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA. 

Video: Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

Environment

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO1, leprosy remains endemic in many countries with approximately 250,000 new cases each year.2 The entire M. leprae genome has been mapped3,4 and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites).5 Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci.5,6,7 Variable number tandem repeat (VNTR)5 analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing7,8,9, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA)10 has been used to study leprosy evolution and transmission in several countries including China11,12, Malawi8, the Philippines10,13, and Brazil14. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS).10 The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions.10 The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory.  ...

Immunology and Infection

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Biology

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

DNA Isolation and Restriction Enzyme Analysis

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.

<h4>Isolating DNA from Cells</h4>

DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na+ ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.

<h4>Quantifying DNA</h4>

Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.

<h4>Restriction Enzymes</h4>

For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.

Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes1.

DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases2. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions3. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects4.

<h4>References</h4>

<ol class="references">
	<li>Chambers, GK, et al. DNA fingerprinting in zoology: past, present, future. Investig Genet. 2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li>Roewer, L. DNA fingerprinting in forensics: past, present, future. Investig Genet. . 2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li>Kirkpatrick, BE and Rashkin, MD. Ancestry Testing and the Practice of Genetic Counseling. J Gen Counseling. 2016, Vol. 26, 1 (6-20).</li>
	<li>Hamburg, MA and Collins, FS. The Path to Personalized Medicine. N Engl J Med . 2010; , Vol. 363, 301-304.</li>
</ol>

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Education

JoVE Lab Manual

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of agarose, and within the gel, charge particles will migrate and separate based on size. The negatively charged phosphates of the DNA backbone cause DNA fragments to move toward the anode - a positively charged electrode.
The video explains the mechanism by which DNA fragments are resolved on an agarose gel, and it provides a step-by-step generalized procedure for how to prepare agarose gels, load DNA samples, run a DNA gel, visualize DNA fragments, and properly dispose of the gel and running buffer after the experiment is concluded.

DNA Gel Electrophoresis: Concept, Procedure, and Applications

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Summary

Explore More Videos

Chapters in this video

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

DNA-based Fish Species Identification Protocol

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Robust 3D DNA FISH Using Directly Labeled Probes

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

DNA Isolation and Restriction Enzyme Analysis

DNA Gel Electrophoresis