DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design.
A custom Entrez gene-based CDF file was used for probe set condensation and annotation (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp).edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp
Primers used for probe synthesis are listed in Table S5.
Bacterial and viral genes used for probe and primer selection are listed in Table 1.
Human Genome U133 Plus 2.0 arrays (Affymetrix) were used for probe hybridization and processed according to recommended protocols.
Oligonucleotide primers used for probe synthesis are listed in Table 1 and probe binding sites are illustrated in Figure 1.
Goat anti-DIG antibody conjugated to alkaline phosphatase (AP) was used for probe detection and NBT-BCIP was used as the substrate for color development.
An isothermal format (Tm = 76°C) [42] and probe length constraint between 50 and 75 bp were used for probe synthesis.
All other constructs used for probe synthesis were full-length cDNA or EST image clones obtained from Open Biosystems (http://openbiosystems.com/) and verified by sequencing.
Two micrograms of total RNA were used for probe synthesis of cy3- and cy5-labeled cRNA, and hybridizations were carried out on an Agilent-based microarray platform.
Our other approach to assess MM tolerance was to hybridize the array to genomic DNA from organisms with varying degrees of relatedness to the four target strains used for probe design.
