PhD Thesis Demeter Zoltan

4.5.5 Nucleic acid sequencing and phylogenetic analysis
Following electrophoresis in a 2 % Low Melting Agarose Gel (Bio-Rad Laboratories,
USA), the amplicons were cut out from the gel, and DNA was extracted with the
QiaQuick Gel Extraction Kit (Qiagen, Germany). Fluorescence-based direct sequencing
was performed in both directions on the amplicons at Biomi Kft. (Gödöllı, Hungary)
and Biogon Kft. (Budapest, Hungary), using ABI 3100 genetic analyzers (Applied
Biosystems, USA). The nucleotide sequences were identified using the BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/). The two direction sequences were compiled
and aligned using the Align Plus 4 software (Scientific and Educational Software, USA)
and Clustal X. Phylogenetic trees were constructed using the nucleic acid sequences of
the Hungarian CDV, FPV and CPV2 strains, as well as sequences of corresponding
lengths acquired from the GenBank (http://www.ncbi.nlm.nih.gov) by neighbor-joining
with two parameters distance matrix (Kimura, 1980), using the Phylip program. The
robustness of the groupings in the neighbor-joining analysis was assessed with 1000
bootstrap resamplings in case of the CDV strains and 100 in case of the parvovirus
strains.
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