PCR, polymerase chain reaction.
PCR, polymerase chain reaction.
11. To inactivate the T4 DNA ligase, heat the samples at 65°C for 20 min.
12. Carry out a second round of DNA double restriction to cleave any restriction site reformed by ligation with 5 U of each restriction enzyme and the corresponding buffer for 15-30 min.
13. Carry out a PCR reaction with the following reagents: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 ^M each dNTPs, 1 ^M of each of the corresponding primers (Table 2), 1 U of AmpliTaq polymerase, and 1 ^L of the restricted-ligated DNA. The final reaction volume is 50 ^L.
14. Perform the PCR reaction in the thermal cycler at the following temperatures: 94°C for 6 min and one cycle and 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, for 30 cycles.
15. Prepare an 8% (w/v) polyacrylamide gel adding 2.66 mL acrylamide/bis solution to 7.34 mL 1X TBE buffer. Add 75 ^L of 10% (w/v) ammonium persulfate and 10 ^L TEMED and let it polymerize for 1 h in a Mini Protean electrophoresis cell.
16. Mix samples and the molecular weight standard with loading buffer and run an electrophoresis with 1X TBE buffer at a constant 150 V for 45 min.
17. Stain the polyacrylamide gel with ethidium bromide (3 ^g/mL) for 15 min, wash the gel with distilled water for 30 min, and photograph under UV transillumination.
18. Interpret the banding patterns visually or with help of a computerized analyzer.
3.4. Computerized Analysis of Fingerprinting Patterns
1. Scan and save images of gels in a TIFF file and then analyze them by GelCompar version 4.0 or superior software (Applied Maths; see Note 5).
2. After conversion and normalization of gels, determine the degrees of similarity of DNA fingerprints by Dice or related coefficients (see Note 8).
3. Generate dendrograms using the UPGMA (unweighted pair group method using arithmetic averages) algorithm.
4. To construct the libraries with the PFGE fingerprints, follow the instructions of the software manufacturer. Such libraries allow the user to identify new eletrophoretic patterns and to incorporate them in the database.
1. N-laurylsarcosine may precipitate when lysozyme and RNAse are added. If this happens, place the reaction product at 50°C in a water bath to dissolve and then warm it at room temperature.
2. PMSF is highly toxic by inhalation. Its use for Proteinase K inactivation could be avoided, but more washing with distilled water is then required.
3. In some Salmonella serotypes, extensive DNA degradation is observed during electro-phoresis. Addition of thiourea to the electrophoresis buffer prevents such events.
4. Several primers are available in the literature for such PCR fingerprinting procedures, and different levels of strain discrimination could be achieved depending on the serotype studied.
5. GelCompar is licenced in the United States and several countries to Bio-Rad laboratories. Other software such as Molecular Analyst run on an Apple platform. The Bio Image Whole Band Analyzer runs on a Unix platform. We have compared and evaluated such software to determine whether results generated by these programs correlated adequately with visual interpretation of DNA patterns (9).
6. The interassay reproducibility of PCR fingerprinting is inherently low owing to the low annealing temperatures and influences of minor changes in the amounts of reagent in the PCR reaction (10). Intra-assay reproducibility is higher, allowing for analysis of a group of isolates in a single gel looking for band differences.
7. Choose the adapters in relation to the restriction enzyme used as shown in Table 1.
8. The software packages try to imitate the automatic band alignments that our eyes perform and usually accomplish it successfully when gels are of good quality. Computerized alignments of the internal control bands in heavily distorted gels are frequently performed inaccurately; therefore such gels should be eliminated from the comparison or repeated.
This work was supported by The University of Basque Country and the Basque Government. A. B. Vivanco is supported by a grant from The University of Basque Country and J. Alvarez by a grant from the Basque Government, Spain.
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9 Rementeria, A., Gallego, L., Quindos, G., and Garaizar, J. (2001) Comparative evaluation of three commercial software packages for analysis of DNA polymorphism patterns. Clin. Microbiol. Infect. 7, 331-336.
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