Biomedical Engineering Reference
In-Depth Information
Single-copy protein-coding gene sequences also accumulate in the public
databases and may be used to complement those of D1/D2 LSU and ITS sequences
in cases where the ribosomal genes do not allow a conclusive species identification.
Such protein-coding genes include the actin gene (
ACT1
) [
55
] , translation elonga-
tion factor 1 alpha (
TEF1
), the mitochondrial cytochrome oxidase 2 gene (
COX2
)
[
211
], and the largest and second largest subunits of the RNA polymerase II gene
(
RPB1, RPB2
) [
212
]. In contrast to the multicopy ribosomal DNA (e.g., D1/D2
LSU, ITS), single-copy nuclear genes may be more difficult to amplify, as the design
of universal primers effective in phylogenetically distant species is not always pos-
sible and as only single primer binding sites are available in a haploid genome in
contrast to hundreds of binding sites for ribosomal genes. Highly variable mito-
chondrial genes (e.g.,
COX2
) bear the possibility of having been subject to a differ-
ent evolutionary path than the nuclear genome, in other words, are prone to potential
horizontal gene transfers across species borders. As databases for complementary
gene sequences are far more restricted than for the D1/D2 LSU, one needs to assure
the existence of reference sequences. Databases that allow searching the available
sequences for specific strains, such as the yeast database of the Centraalbureau voor
Schimmelcultures, The Netherlands (
www.cbs.knaw.nl/yeast/BioloMICS.aspx
)
and
the StrainInfo portal (
www.straininfo.net/
), Ghent University, Belgium, may be
used for the selection of complementary sequencing targets. The comparison of a
query sequence with reliable type strain sequences is essential, as type strains are
the only valid taxonomic reference for a species. Sequence alignments outside the
commonly consulted BLAST (Basic Local Alignment and Search Tool) tabulated
results are helpful, because type strain sequences are often not recognizable from
the sequence entry title line. It is recommended to include type strain sequences of
the phylogenetically most closely related species in these alignments to confirm the
differentiation of the species in question by the given DNA region.
While the so far discussed methods use genetic information of few or single loci,
the techniques commonly known as DNA fingerprinting exploit genetic information
that is distributed throughout the genome. A large variety of protocols exists and the
more reproducible among them are based on the specific binding of PCR primers to
mini- or microsatellite sequences in contrast to arbitrary binding realised in RAPD-
PCR. The primer most frequently applied to sourdough yeasts was derived from a
ubiquitous minisatellite sequence found in the protein II gene of the bacteriophage
M13 [
213
]. The primer referred to as M13 results from a consensus sequence of 12
partially incomplete repeats. After its use in a PCR assay as the single primer and
visualization of the PCR reaction products by agarose gel electrophoresis, usually
species-specific banding profiles based on the different lengths of amplifiable
sequences enclosed or flanked by M13 minisatellites are observed. The important
influence of experimental factors such as the DNA extraction method and PCR and
electrophoresis parameters on the resulting profiles implies the need to include type
strains for ideally side-by-side-comparisons if a complete identification is to be per-
formed. However, PCR-fingerprinting without type strains may be used to group
larger numbers of isolates and to select those that are representative of each group
for identification by DNA sequencing [
40
] .
