1994 the primer sets were synthesized by eurogentec

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(1994). The primer sets were synthesized by Eurogentec (Seraing, Belgium). Amplification was performed in a Bio-Rad Gene Cycler TM , version 1.5. (Bio-Rad, Hercules, CA, USA). Each 30 l l of reaction mixture contained 1X PCR buffer (10 mmol l ) 1 Tris–HCl, 50 mmol l ) 1 KCl and 0 Æ 1% Triton X-100, pH 9 Æ 0), 200 l mol l ) 1 each of dATP, dCTP, dGTP and dTTP (Promega, Madison, WI, USA), 2 mmol l ) 1 MgCl 2 , 60 pmol of each primer, 1 Æ 0 U of Taq polymerase (Promega), and 4 l l of lysed cell suspension. A negative control, consisting of the same reaction mix- ture but without DNA template, was included in each amplification procedure. The thermal cycling procedure was that used by Louws et al. (1994). Subsequently, the PCR amplification products were separated by gel electro- phoresis on a 2 Æ 5% agarose (Seakem-Cambrex, Rockland, ME, USA) gel in 1X Tris-borate–EDTA (TBE) buffer (80 mmol l ) 1 Tris-borate, 89 mmol l ) 1 boric acid, 2 mmol l ) 1 EDTA; pH 8 Æ 0) at 60 V cm ) 1 for 3 h, stained with ethidium bromide, visualized with a UV transillumi- nator, and photographed with Polaroid type 55 film (Polaroid, Cambridge, MA, USA). The PCR amplifica- tions were performed in duplicate. The method of Smith et al. (1995) was used for analysis. The clearly resolved bands present in both amplification gels were scored and recorded to build up a binary matrix. Similarity coeffi- cients for all pairwise combinations were determined by using the Dice (1945)’s coefficients and were clustered by unweighted pair group method with arithmetic means (UPGMA), using the ntsys - pc software (Exeter Software, New York, NY, USA), version 2.11j (Rohlf 2000). Pheno- grams were constructed with the tree display option ( tree ). A cophenetic value matrix was calculated using the coph option and compared with the original similar- ity matrix using the mxcomp option to test the goodness- of-fit of the cluster analysis. ARDRA analysis The basic ARDRA technique described by Vaneechoutte et al. (1992) was utilized. The DNAs coding for the 16S rRNAs of 40 E. amylovora strains (see Table 1) were amplified with primers P0 (5 ¢ -GAGAGTTTGATCCTGG- CTCAG-3 ¢ ) and P6 (5 ¢ -CTACGGCTACCTTGTTACGA- 3 ¢ ). These primers were designed by Grifoni et al. (1995) on the basis of the conserved bacterial sequences at the 5 ¢ - and 3 ¢ -ends of the 16S rRNA gene and allowed the amplification of almost the entire gene. Each 50- l l (final volume) of PCR mixture contained 6 l l of lysed cells sus- pension, Promega Taq buffer (1 Æ 5 mmol l ) 1 MgCl 2 ), each deoxynucleoside triphosphate at a concentration of 200 l mol l ) 1 , 36 pmol of each primer and 1 Æ 5 U of Taq DNA polymerase (Promega). The reaction mixtures were incubated in a Bio-Rad Gene Cycler TM , version 1 Æ 5 at 95 Ŷ C for 2 min and then subjected to 35 cycles at 95 Ŷ C for 30 s, 30 s of annealing (60 Ŷ C for the first five cycles, 55 Ŷ C for the next five cycles, and 50 Ŷ C for the last 25 cycles), and 72 Ŷ C for 4 min. Finally, the mixtures were incubated at 72 Ŷ C for 10 min and then at 60 Ŷ C for 10 min. Five microlitres of each amplification mixture was analysed by 1 Æ 5% agarose (Seakem) gel electrophor-
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  • Spring '08
  • Devartanian
  • DNA, DNA sequencing, Restriction enzyme, Greece Greece Greece Greece Greece Greece Greece Greece Hungary Hungary Hungary Hungary Hungary Hungary Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy USA USA Italy Italy Italy Italy, E. amylovora

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