The overall goal of this procedure is to develop a distinguishing DNA fingerprint of mycobacterium lere, the non cultivable bacterium that causes leprosy directly from clinical specimens. M lri bacterial, DNA extracted from patient biopsies is amplified by a multiplex PCR using fluorescent primers specific to the variable number tandem repeat or VNTR loci. Once the DNA has been amplified, capillary electrophoresis is performed for fragment length analysis.
The data are analyzed to determine the number of short tandem repeat DNA segments at each locus. Because different strains of EPRI exhibit different numbers of tandem repeats at each locus. Data from multiple loci produces unique DNA fingerprints and a database of patient's strain types can be generated using this technique.
The population structure, evolution and transmission of leprosy between patients in defined communities and across the globe can be characterized, ultimately providing genetic tools and evidence for the elimination of leprosy. This method can be used for tracing the spread of leprosy, for identifying clusters of recent infections and at-risk individuals and for measuring the efficacy of controlled programs. Demonstrating the procedure will be we Lee and Ron Jensen, researchers from my lab and Jason Rivers from our instrument core facility Extract, DNA from clinical biopsies using a standard KaiGen DNA extraction kit.
Once the DNA has been obtained, set up a new work area inside a clean PCR cabinet. To avoid any DNA contamination wipe down the cabinet and a set of pipettes with 70%ethanol and subject the cabinet and pipettes to UV light for 15 minutes. Once the cabinet is clean, the multiplex PCR can be set up for each multiplex PCR four to five primer pairs in which one primer per pair is fluorescently labeled.
Must be mixed. Use 10 micromolar primer stocks prepared in te buffer to make the primer mixes mix equal amounts of all the forward or upper primers in an einor tube so as to achieve a final concentration of two micromolar. For each primer, add TE buffer if needed.
In another einor mix all the reverse or lower primers the same way. Once the primer mixes have been made, prepare the PCR MES mix. Make a plate map labeled with all samples and controls per sample.
The master mix should contain 12 microliters of KaiGen multiplex PCR mix, two microliters of each primer pool and two microliters of Q solution. Be sure to make enough master mix to include controls and to account for pipetting losses. Briefly centrifuge the master mix.
Next aliquot 18 microliters of master mix into each well of a PCR plate. To avoid contaminating the DNA samples, change gloves, wipe down and disinfect a separate biosafety cabinet. As before, once the hood has been decontaminated, transfer the plate to that hood.
Then add two microliters of DNA sample to each well containing master mix. Be sure to use aerosol prevention pipette tips to avoid cross-contamination. Carefully seal the PCR plates and centrifusion briefly to mix the contents.
Then place the reaction plate in the thermocycler and start the PCR program for submitting samples. For FLA, prepare the form amide size, master mix, and add diluted PCR products in a clean einor tube combined 12 microliters of high dfor amide solution and 0.3 microliters of gene scan, 500 lid sizing standard per sample to be tested. Aliquot 12.3 microliters of this solution per well into a 96 well optical quality reaction plate.
Once the PCR is complete, the plate is centrifuged. Be sure that the position of each sample is recorded on the plate map. Then in a separate 96 well plate aliquot 59 microliters of nuclease free water per well and add one microliter of each PCR reaction per well to dilute the amplified DNA at one to 60.
Once the DNA has been diluted, use a multi-channel pipette to transfer one microliter of each diluted PCR sample into the corresponding well of the plate containing the form amide loose solution. Once the program is complete, the samples may be tested by standard agros gel electrophoresis to confirm the DNA fragments of the predicted sizes have been amplified at this point. The samples are prepared for analysis by fluorescent capillary electrophoresis using an FLA genetic analyzer prior to analysis, add a septum to the plate and centrifuge briefly to remove air bubbles and knock the sample into the well.
Thefor IDE will chemically denature the strands so there's no need to heat denature. Place the plate in a loading tray. Evaluate the labeled PCR product using capillary electrophoresis such as ABBI'S genetic analyzer 31 30 xl.
Analyze the samples using a 50 centimeter capillary filled with pop seven polymer as the separating medium. The DNA migrates toward the end of the capillary where a laser and detector measures the fluorescent labels open the analyzer's collection software. The analyzer will need to be calibrated to detect all dyes used prior to running the samples, including the size standard the dset used for these samples is ABIs G five Dissect samples are electro kinetically injected at 1.6 kilovolts for 15 seconds.
Separate the fragments by applying 15 kilovolts for 1800 seconds. The total runtime for each run is about 45 minutes. Once the run has finished, create data files for each sample.
The files are placed in a plate folder by the analyzer's collection software in a predetermined location. Files are in FSA format. FLA data can be analyzed using peak scanner software, which is freely available on the Applied Biosystems website.
To analyze data, click start new project, followed by add files. Load the selected data files into the program, including the positive and negative controls. Select and analyze the loaded files.
The results are plotted as an FLA chromatogram. The sizing standards appear as orange peaks, whereas the colored peaks represent the bacterial DNA applicants for each locus analyzed in the sample, click on a peak to determine its base pair size. Due to DNA polymerase slip strand synthesis, a family of peaks of the same color will be detected for some microsatellite loci.
In these cases, the selected peak is usually the one of largest amplitude. Compare the fragment sizes in each sample to the positive control size to determine how many tandem repeat segments are present for each ale l tested and record the data into an Excel spreadsheet for further analysis. In this figure, the DNA fingerprint of isolates from seven patients belonging to three different families from the same village are shown and the number of tandem repeats for each polymorphic locus analyzed is indicated.
These data suggest that two members of family be are infected with the same strain, whereas one member is infected with a distinct strain. Members of families C are likely infected with the same strain. As are members of family A.After watching this video, you should have a good understanding of how to routinely include multiplex PCR and fragment length analysis to obtain V-N-T-R-D-N-A fingerprints for leprosy patients presenting in clinics.
These are novel molecular tools to complement conventional epidemiology for leprosy disease control.
