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The results prompt that species-specific probes must be designed in regions of the gene unique to T. equi.
Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.
Primers were specifically designed in regions of low homology with the other UCP genes and flanking one or more introns.
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All primers were designed in regions free of known SNPs.
In addition, nine probes were designed in regions that showed no evidence of DNaseI hypersensitivity.
The 5' end of the 28S rRNA gene was amplified via PCR, using primers designed in conserved regions of the ribosomal cistron of molluscs (Additional file 5).
With the aid of this alignment and the annotation of H. annuus, primers were designed in conserved regions of genes.
A total of 82 primer pairs were designed in flanking regions of the randomly selected target eSNPs using the Primer3 program.
Two sets of primers specific for each virus, were designed in conserved regions of the viral genome for their use in the one-step real-time RT-PCR.
Three compatible sets of primers, specific for each virus, were designed in conserved regions of the viral genomes for use in a one-step (single tube) RT-PCR assay.
To confirm this assignment, the genomic DNA of CS was amplified by primer pairs designed in different regions of the sequence; several different amplification patterns displaying from one to four fragments were obtained by electrophoresis in 2% agarose gel (data not shown).
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