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Amplification, cloning, and sequencing
The ITS1±5.8S±ITS2, 28S and 18S ribosomal regions of
the helicosporidial DNA were amplified with a mixture of
Taq DNA polymerase (Promega) and Pfu polymerase
(Stratagene), using the primers TW81 and AB28 for the
ITS±5.8S (Curran et al., 1994) and NL-1 and NL-4 primers
for the 28S (Kurtzman & Robnett, 1997). Two primer sets,
designed from consensus regions of selected fungal, algal
and protozoan sequences downloaded from GenBank, were
used to amplify the 18S region. Several series of primers, also
designed from consensus regions of selected fungal, algal
and protozoan genes, were used to amplify partial sequences
of the actin and β-tubulin genes by PCR. DNA was excised
from agarose gels, extracted with the QiaxII gel extractionkit (Qiagen) and sent to the Interdisciplinary Center for
Biotechnology Research (ICBR) at the University of Florida
for sequencing.
View list of primers
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