Rep-PCR
The use of repetitive element PCR fingerprinting is molecular biology based method very suitable for rapid grouping and tentatively identification of microorganisms. Eukaryotic and prokaryotic DNA contains so-called repetitive DNA elements distributed more or less randomly over the genome. In rep-PCR primers that anneals to these repetitive elements are used. The PCR-products are separated using agarose gel electrophoresis and a species (sometimes strain)-specific pattern is obtained. These patterns can be analysed using e.g. BioNumerics. Isolates with similar patterns (i.e. belonging to the same species) will cluster together. Full identification can be achieved by e.g. sequencing a limited number of isolates from each group within the cluster. Various rep-PCR-primers have been developed. The primer GTG5 (5'GTG GTG GTG GTG GTG 3') seems to be very suitable for grouping of LAB and yeast at the species level, but other primers may prove better depending on the specific task (use the same over all approach, just change primers and possibly annealing and elongation temperature).

Note: Rep-PCR is only suitable for grouping isolates that have been partially characterised (e.g. catalase, Gram-reaction and microscopy). A suitable group for Rep-PCR would for instance be Gram-positive, catalase-negative rods and cocci originating from MRS (i.e. presumptive LAB).

Protocol, rep-PCR using the GTG5-primer:
1. Extract DNA using Instagene (Bio-Rad) following the instructions of the manufacturer. Use only ½ volume of the Instagene mixture (100 μl instead of 200 μl).

2. Rep-PCR.
Rep-PCR mixture (pr. sample):
Compound Volume
PCR-buffer (10-) 2.5 μl
dNTP-mix (1.25 mM) 4.0 μl
MgCl2 (25 mM) 1.5 μl
Primer (GTG5, 5 pmol/μl) 4.0 μl
Formamide* 0.25 μl
BSA (Bovine Serum Albumin, 0.1 mg/ml)* 0.25 μl
Sterile MilliQ-water 10.8 μl
Taq polymerase 0.2 μl
DNA 1.5 μl
Total 25 μl
* Both can be omitted. If omitted adjust volume of water to keep total volume constant at 25 μl.

NOTE: It is very important the DNA extracted using Instagene is whirli-mixed and subsequently centrifuged (13000 G, 3 min.) before use (this step MUST be repeated every time the DNA is used!!).

Include negative control (water instead of DNA) to check for impurities.

Use the following thermo cycling programme:
94 °C 5 min.
94 °C 30 sec.
45 °C 60 sec. 30 cycles
65 °C 8 min.
65 °C 16 min.
4 °C 24 hours

3. Separate PCR-products by agarose gel electrophoresis: Prepare a 1.5 % agarose gel in 1-TBE. Fill running chamber with 1-TBE (10-TBE = 108 g Trisbase/l, 55 g boric acid/l and 40 ml of 0.5 M EDTA, pH 8.0). Mix PCR-products with 5 μl of Loading Dye. Load 12 μl of sample. Load 4 μl of marker into every tenth well (e.g. on a 30-well gel load marker in well 1, 10, 20 and 30). Run electrophoresis (120 V, 5 h).

4. Stain gel with ethidium bromide.

5. Document gel using digital camera.

6. Carry out cluster analysis using BioNumerics (see guide in separate document). Use DICE alternatively Pearson. Use UPGMA as the clustering algorithm.

7. Good luck.

References

Andrade, M. J., Rodriguez, M., Sánchez, B., Aranda, E., & Córdoba, J. J. (2006). DNA typing methods for differentiation of yeasts related to dry-cured meat products. International Journal of Food Microbiology, 107, 48-58.
Gevers, D., Huys, G., & Swings, J. (2001). Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiology Letters, 205, 31-36.
Nielsen, D. S., Schillinger, U., Franz, C. M. A. P., Bresciani, J., Amoa-Awua, W., Holzapfel, W. H., & Jakobsen, M. (2006). Lactobacillus ghanaensis; A novel motile lactic acid bacteria isolated from Ghanaian cocoa fermentations. International Journal of Systematic and Evolutionary Microbiology., Submitted for publication-
Nielsen, D. S., Teniola, O. D., Ban-Koffi, L., Owusu, M., Andersson, T., & Holzapfel, W. H. (2007). The microbiology of Ghanaian cocoa fermentations analysed using culture dependent and culture independent methods. International Journal of Food Microbiology., 114, 168-186.

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