Pcr amplification was performed in a bio rad

Info icon This preview shows pages 6–7. Sign up to view the full content.

View Full Document Right Arrow Icon
50 pmol of each primer. PCR amplification was performed in a Bio-Rad GenCycler TM , version 1.5 and included a denaturation step at 92 Ŷ C for 1 min, followed by 35 cycles at 92 Ŷ C for 1 min, 52 Ŷ C for 1 min and 72 Ŷ C for 1 min with a final DNA extension step at 72 Ŷ C for 2 min. Aliquots of 10 l l of the PCR amplification were analysed on 1% ag- arose gel (Seakem) for 30 min at 100 V cm ) 1 , in Tris-acet- ate buffer. A 100-bp DNA ladder (Promega) was used as a size marker. For the RFLP analysis, aliquots of 40 l l of PCR products were purified by ethanol precipitation. The purified DNAs were resuspended in 16 l l of sterile water. Eight microlitres of aliquots were used for digestion with 5 U Msp I and 5 U Sau 3A restriction enzymes (Promega) for 2 h in a total of 15- l l volume at 37 Ŷ C. The samples were analysed on 3 Æ 0% agarose gel (Seakem). Short-sequence DNA repeats analysis The SSRs analysis was performed for 61 E. amylovora strains. For detecting the presence of SSR units, 10 l l of aliquots of the pEA29 PCR fragment were amplified with primer RS1 (5 ¢ -ACCTCAGTGCGATTACAG-3 ¢ ) and RS2c (5 ¢ -GTCCCATTCTGTGTTAAG-3 ¢ ) (Kim and Geider 1999). The products were analysed on 8% polyacrylamide gel. The number of SSR units was established following the procedure described by Ruppitsch et al. (2004). Briefly, the A/B PCR fragment was amplified by the use of primer sB (5 ¢ -TGTAAAACGACGGCCAGTGGGCAAATACTCGGAT- T-3 ¢ ) and primer sA (5 ¢ -CAGGAAACAGCTATGACCC- GGTTTTTAACGCTGGG-3 ¢ ) (MWG-Biotech) containing the M13 recognition sequence for subsequent DNA sequen- cing analysis. The amplified DNA fragment was ethanol precipitated and sequence analysis was performed by cycle sequencing (SequiTherm Excel II Cycle Sequencing kit; Epicentre, Madison, WI, USA) with fluorescent-labelled primers M13 univ. (5 ¢ -TGTAAAACGACGGCCAGT-3 ¢ ) and M13 reverse (5 ¢ -CAGGAAACAGCTATGACC-3 ¢ ) (MWG-Biotech) using an automated DNA sequencer Licor 4200S (LI-COR, Lincoln, NE, USA) according to the manufacturer’s instructions. Results Repetitive sequences PCR analysis The genetic relationship within E. amylovora strains was assessed using rep- PCR and ERIC, REP and BOX primer sets. Reproducible DNA fingerprints were generated from total DNA of all strains listed in Table 1. REP and ERIC primers resulted more discriminative than BOX in point- ing out variability within E. amylovora strains and are dis- cussed herein. A cophenetic value of >0 Æ 91 and 0 Æ 92 was determined for the two similarity matrix, respectively, indicating a high goodness-of-fit for the cluster analysis. UPGMA analysis was performed by combining the data obtained from REP-PCR and ERIC-PCR. A total of 24 clearly resolved bands were selected for composing the binary matrix. Representative genomic fingerprints are shown in Fig. 1 and the corresponding dendrogram is shown in Fig. 2. The majority (89 of 93) of E. amylovora strains showed the same DNA fingerprint profile. Diver- sity was ascertained for four strains, namely PD 2915, iso- lated from Amelanchier sp. and NCPPB 2292, NCPPB 2293 and PD 103, isolated from Rubus spp. E. amylovora
Image of page 6

Info icon This preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Image of page 7
This is the end of the preview. Sign up to access the rest of the document.
  • 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

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern