Rezumatul tezei de doctorat - USAMV Cluj-Napoca

We used a comparatively mixture of two primer pairs, in the following combination:
ITS1 with ITS4 and ITS4 with ITS5. ITS1 primer is attaching at the 3’ end of the 18S rDNA
gene and ITS4 primer is at the 5’end of the 28S rDNA gene (after Paul B. And al., 2004).
The last combination of primers is used to amplify the region of the rDNA repeat unit that
includes the two non-coding regions designated as the internal transcribed spacers ITS1 and
ITS2, and the 5,8S rDNA gene (Llanos Frutos and al., 2004).
I.3.8. Molecular techniques used in the experiment
I.3.8.1. PCR amplification of Saprolegnia samples
In our experience we used ITS1 and ITS4, ITS4 and ITS5 primer pair combinations
(White and al., 1990), which amplify ITS1 region, 5,8S rRNA gene and ITS2. The
amplification reactions were individually performed, in a final volume of 25 µl, with an
Eppendorf thermocycler.
The amplification cycling parameters were: initial denaturation at 95°C for 3 minutes,
followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute,
and extension at 72°C for 1 minute, with a final extension at 72°C for 7 minutes. The
amplification products were separated by electrophoresis in 1,5% agarose gels, in 1x TBE
buffer, for 60 minutes at 70V. The DNA was stained with Sybr Safe (Invitrogen) and
visualized under UV light. The gel was photographed and the image recorded on the
machine (Biorad).
The lengths of amplification products were estimated by comparison to a DNA
Ladder Low Range (700 bp) or 50 bp (Fermentas) (adapted after Ristaino and al., 1998).
We tested another amplification method, which was performed in an Eppendorf
thermocycler, after the following protocol:
- initial denaturation: 5 minutes at 95°C
- 35 cycles: - denaturation 30 seconds at 95°C
- annealing 30 seconds at 48°C
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