MLP JPH203 molecular weight analysis using capillary electrophoresis was modified from Botterel et al.. Alleles were amplified in a multiplex PCR in a 50 μl final volume containing 20 ng DNA, 1X PCR-Buffer II (Applied Biosystems, Madrid, Spain), 0.2 mM of each deoxynucleotide triphosphate, 5 mM of MgCl2, and 0.15 μM of each primer and 1U of AmpliTaq Polymerase (Applied Biosystems). Sense CDC3 primer was labelled with 4, 7, 2′, 4′, 5′, 7′-hexachloro-6-carboxyfluorescein (HEX), EF3 antisense primer with 6-carboxyfluorescein (FAM)
and HIS 3 sense primer was labelled with 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1.4-dichloro-6-carboxyfluorescein selleck chemicals (NED). Primers were synthesized by Sigma-Aldrich (Sigma-Aldrich, Madrid, Spain). PCR reactions were performed in a GeneAmp PCR system 9700 (Applied Biosystems). The cycling conditions included a first step for preincubation (activation of the enzyme) and denaturation of the DNA template at 95°C during 5 minutes. Next steps consisted in an amplification program of 30 cycles as follow: denaturation at 95°C for 30 s, annealing at 55°C for 30 PRT062607 nmr s and extension at 72°C for 1 min with a final extension step of 7 min at 72°C. To assess the size of the fragments, 1 μl of the
PCR products was added to 9 μl of Formamide Hi-Di (Applied Biosystems, Madrid, Spain) and 1 μl of the internal size standard ROX 500 (Applied Biosystems, Madrid, Spain). Capillary electrophoresis was run using the ABI 3730 XL (Applied Biosystems, Madrid, Spain) sequencer. Fragment size for the different alleles was calculated with GeneMapper version 3.0
(Applied Biosystems, Madrid, Spain). In addition, a HRM-based analysis was performed using singleplex PCRs with each pair of primers without any modification of the reaction conditions. Control population was selected based on MLP results. Strains included as control were: CL 7484, CL 7498, CL 7504, CL 7513, CL 7694, ATCC 64548 and ATCC 64550 (Figure 1). Seven different genotypes for the three markers were chosen (Figure 1). Figure 1 Difference plots for the normalized and temperature shifted melting curves for microsatellite from control population and patient strains. A) 17-DMAG (Alvespimycin) HCl CDC3 marker; B) EF3 marker and C) HIS3 marker. After PCR, HRM analysis was performed in a LightCycler 480 system (Roche, Madrid, Spain). To obtain the HRM curves, 1 μl of LightCycler® 480 ResoLight Dye (Roche, Madrid, Spain) was added to PCR products and the reactions were incubated at 95°C 1 min, followed by a renaturation step of 40°C for 1 min. Melting curves were generated by ramping from 65° to 95° at 0.02°C/s, 25 acquisitions/°C. HRM curves were plotted using the automated grouping option provided by the software and by manual editing for each microsatellite marker. Normalization conditions for each microsatellite marker are shown in Table 4.