Thermal cycling consisted of a denaturation step (2 min @ 95°C) and 18 high-stringency step-down cycles followed by 40 relaxed-stringency fluorescence acquisition cycles.
For loci used for either map, the stringency was stepped down and the map locations were compared.
For the generic probe, three stringency washing steps were performed using 2.0× RiboWash (Ventana Medical Systems; equivalent to 2.0× saline sodium citrate) each for 4 min at 52°C.
The protocol for the vertebrate RLB was the same as for the bacterial RLB except the prehybridization wash, hybridization and high stringency wash steps were all conducted at 62°C.
Following hybridization, the microarray slides were subjected to three-step stringency washes (2X SSC + 1% sarcosyl, 2X SSC, 0.2X SSC).
The first round of adaptive clustering was conducted at the cut-off of 10−15, and the cut-off was reduced to 10−33, thereby increasing the stringency, in 6 steps, to split non-linear contigs.
Starting with a stringent cutoff, the joining was repeated with relaxed stringency in each successive step.
It is possible to relax the stringency of the filtering step in further iterations of the classifier to identify euchromatic repeats that do not resemble genome typical repeats.
PCR reactions were performed with high stringency: an initial denaturation step of 2 min at 94°C was followed by amplification for 25 (B2M) or 35 (CAGR1 CAG- or CAG+) cycles (30 sec at 94°C, 30 sec at 63°C, 45 sec at 72°C), and a final extension for 7 min at 72°C.
Bar charts show the number of NCO GC detected per offspring plant using different marker set generated with different filter stringencies in each subsequent step (top to bottom).
Due to the exact primer-template match for the primer pairs used in the second and third PCR steps, high stringency cycles were employed in these two latter stages (details shown in Table 2).
