Name: dna fingerprinting of mycobacterium leprae strains using variable number tandem repeat (vntr) - fragment length analysis (fla)
Uploaded: 2011-07-15T13:00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA at JoVE.com

Funding was provided by NIH/NIAID grant RO1-AI-63457 and ARRA grant supplement RO1-AI-63457 S1. We acknowledge the contributions of all current and past members of the laboratory group and collaborators.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+leprosy+for+control+programmes.">Chemotherapy of leprosy for control programmes.</a> Technical Report Series. 675, 18-22 (1982).</li><li>Cole, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+gene+decay+in+the+leprosy+bacillus.">Massive gene decay in the leprosy bacillus.</a> Nature. 409, 1007-1011 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leproma+Web+Server.">Leproma Web Server.</a> Leproma Web Server [Internet]. , (2004).</li><li>Groathouse, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Polymorphic+Loci+for+Molecular+Typing+of+Strains+of+Mycobacterium+leprae.">Multiple Polymorphic Loci for Molecular Typing of Strains of Mycobacterium leprae.</a> J. of Clinical Microbiology. 42, 1666-1672 (2004).</li><li>Young, S. 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+of+Short+Tandem+Repeat+Sequences+to+Study+Mycobacterium+leprae+in+Leprosy+Patients+in+Malawi+and+India.">Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India.</a> Public Library of Science: Neglected Tropical Diseases. 2, E214-E214 (2008).</li><li>Monot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Variable-Number+Tandem+Repeats+Appropriate+for+Genotyping+Mycobacterium+leprae.">Are Variable-Number Tandem Repeats Appropriate for Genotyping Mycobacterium leprae.</a> J. of Clinical Microbiology. 46, 2291-2297 (2008).</li><li>Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Variable+Number+Tandem-Repeat+Genotyping+for+Mycobacterium+leprae+Clinical+Specimens.">Rapid Variable Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens.</a> J. of Clinical Microbiology. 47, 1757-1766 (2009).</li><li>Weng, X. i. a. o. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Distribution+of+Mycobacterium+leprae+Genotypes+in+a+Region+of+High+Leprosy+Prevalence+in+China:+a+3-Year+Molecular+Epidemiological+Study.">Identification and Distribution of Mycobacterium leprae Genotypes in a Region of High Leprosy Prevalence in China: a 3-Year Molecular Epidemiological Study.</a> J. of Clinical Microbiology. 45, 1728-1734 (2007).</li><li>Weng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+leprosy+in+Qiubei+County,+Yunnan,+China:+Insights+from+an+eight+year+molecular+epidemiology+investigation.">Transmission of leprosy in Qiubei County, Yunnan, China: Insights from an eight year molecular epidemiology investigation.</a> Infection, Genetics and Evolution. , (2010).</li><li>Sakamuri, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-Based+Molecular+Epidemiology+of+Leprosy+in+Cebu,+Philippines.">Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines.</a> J. of Clinical Microbiology. 47, 2844-2854 (2009).</li><li>Fontes, A. N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+Mycobacterium+leprae+isolates+from+Brazilian+leprosy+patients.">Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients.</a> Leprosy Review. 80, 302-315 (2009).</li></ol>

No conflicts of interest declared.

The collection of skin samples from leprosy patients requires skilled clinicians or technicians working at skin clinics. Laboratory workers handling these samples must take great care to wear lab coats, gloves and protective eye wear and to work in a bio-safety cabinet when handling infected samples of human or armadillo tissue. Disinfection of surfaces and tools is also critical. Working in a clean, sterile bio-safe cabinet is important for avoiding contamination of DNA samples.
DNA extraction has become relatively easy thanks to the development of extraction kits from companies like Qiagen. Directions must be followed carefully. All samples should be kept cold when not in use. Avoid repeated, extreme temperature changes for samples.
DNA primers used in this work can be ordered from various companies which have been cited in the list of literature references at the end of this paper. Care should be taken to work with primers in a DNA free environment in order to avoid contamination. Primers come as dry powder and must be mixed with TE and diluted to a 100&mu;M concentration, then separated into smaller quantities (Figure 2a). Working solutions of primers are further diluted to 10&mu;M concentrations. Again, do not use DNA samples in areas/hoods where primers are used and prepared.
PCR products should also be kept cold when not in use.
A 3% agarose gel preparation will give better DNA band separation, but takes longer to run. For purely qualitative purposes, 2% is usually sufficient. Ethidium bromide solution used for staining the DNA in agarose gels is a highly active genotoxin that is absorbed through the skin. Handle this solution and gels stained with it using great care and always being sure to wear gloves. Wash hands thoroughly after handling any DNA samples or ethidium bromide materials. Dispose of ethidium bromide staining solution, wash and gels according to institutional guidelines. Gels are not routinely required; this step can be eliminated once FLA methods have been established. It is time and reagent consuming.
The formamide solution used in preparing samples for FLA is also highly toxic and should be handled with care. Wash hands after its use. Dispose of it following institutional guidelines.
Reading the results of FLA using Peak Scanner software can be challenging. A basic set of allele calls (number of tandem repeats) has been developed at CSU (Tables 4-7). (It should be noted that Tables 4-7 may not be all inclusive. As other strains of M. leprae are studied, alleles with copy numbers outside the ranges listed may be found.) One particular challenge involves 'stutter' peaks. These are families of peaks, especially of STRs that only involve 2-3 base pair repeats, such as (TA)10. Sometimes selecting the correct peak to read is difficult (Figure 7). In this figure, the peak in the positive control at 190 bp is the main peak. Peaks lower in height that are 2, 4, or even 6 base pairs larger or smaller than the main peak are called 'stutter peaks.' A-tailing can also cause confusion. A-tailing is described earlier in the text and in Figure 8. Finally, if PCR products are highly abundant in the samples, peaks may appear with a double spike. In this case, read the center of the peak form. (See Figure 6, Patient 6.)
Data management can be a formidable task for this type of work. It is important to record all pertinent information for all experiments such as: date of a task or procedure, operator, strip/plate maps, FLA orders, PCR conditions and recipes, dates of gels and photographs, storage temperatures, DNA template dilutions, FLA electronic file storage locations, etc. Good organization and data management can save hours of time spent looking for specific pieces of information in the future.
The laboratory work described here has been going on for several years at Colorado State University and at other locations around the world. The large picture of what all of the collected data means and how it may be of future use is beginning to emerge. Figures 10A and 10B demonstrate how these DNA fingerprints can be used to distinguish different M. leprae strains between countries (10A) or even between families (10B). Family and community linked cases have been shown to carry M. leprae of similar or identical VNTR strain types. The hope is that it may be possible to gain further insight into the mode(s) of leprosy transmission so that an early detection system of transmission networks may be developed for those people most at risk, and that curative drug therapy may begin before permanent neurological and dermatological damage is done.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>DNeasy Blood and Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Multiplex PCR Kit</td>
			<td>Qiagen</td>
			<td>206143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNA Primers</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose Gel</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5x or 6x Gel Loading Buffer</td>
			<td>New England Biolabs</td>
			<td>B7021S</td>
			<td>Recipes available to make your own*</td>
		</tr>
		<tr>
			<td>Ethidium Bromide solution</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>Dilute from con.</td>
		</tr>
		<tr>
			<td>Hi-Di Formamide Solution</td>
			<td>Applied BioSystems</td>
			<td>4311320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gene Scan -500 LIZ</td>
			<td>Applied BioSystems</td>
			<td>4322682</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<a href="http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html">* http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html</a>
<table border="1">
	<tbody>
		<tr>
			<th>Equipment</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>2 Refrigerators</td>
			<td>4&deg;C: 1 for DNA samples, 1 for primers and Multiplex reagents</td>
		</tr>
		<tr>
			<td>1 microwave oven</td>
			<td>Or suitable way for heating agarose and water to make gels</td>
		</tr>
		<tr>
			<td>1 autoclave</td>
			<td>Or pressure cooker for sterilizing materials</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Thermocycler</td>
		</tr>
		<tr>
			<td>2 sets of pipettes</td>
			<td>0.5-10 &mu;l, 10-100 &mu;l, 20-200 &mu;l, 1000 &mu;l 1 set for primers, 1 set for DNA</td>
		</tr>
		<tr>
			<td>Submarine gel electrophoresis box</td>
			<td>With forms and combs for preparing gels.</td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>With cables for connection to gel box</td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>For Eppendorf tubes</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>For Eppendorf tubes/strips</td>
		</tr>
		<tr>
			<td>Capillary electrophoresis system (genetic analyzer)</td>
			<td>Or access to an institution that can perform this analysis</td>
		</tr>
		<tr>
			<td>Plastics</td>
			<td>Disposable aerosol pipette tips, Eppendorf tubes, 8-well strips, 96-well plates, some of optical quality</td>
		</tr>
		<tr>
			<td>Computer with Internet connection</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA at JoVE.com

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.

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.  ...

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