Genemarker software

Genomic DNA was extracted from peripheral leukocytes and formalin-fixed, paraffin-embedded pancreatic tissue, using standard procedures. The coding regions and intron/exon boundaries of the KCNJ11 and ABCC8 genes were amplified by PCR using DNA extracted from leukocytes. The PCR products were Sanger sequenced, and the traces were compared to published sequences using Mutation Surveyor Software.
Seven microsatellite markers spanning chromosomes 11p15.5-11p15.1 were amplified in DNA extracted from the formalin-fixed, paraffin-embedded pancreatic tissue and leukocyte DNA from the patient and both parents. The resulting data were analysed using Genemarker software (Soft Genetics, State College, Pa., USA).

For cell lines not expressing a known point mutation at the ALK locus, tumor cell identity was verified using STR analysis. Genotyping was conducted using an STR-based assay developed by Silicon Biosystems as a multiplex PCR, compatible with Ampli1 WGA digest, with 11 loci detected across 4 fluorescence channels with capillary electrophoresis, using as input only a 1 μl aliquot of the WGA product from single cells, generated as described above. STR reaction mix was added to each sample as well as to a no-amplification negative control. Samples were incubated in a thermal cycler using the following cycles: one 15 min cycle at 94°C and then 32 cycles with each cycle including 50 s at 94°C, 50 s at 60°C, and 1 min at 72°C. The STR call-rate was calculated by dividing the number of alleles detected for each single cell by the number of alleles expected by the STR profile obtained from genomic DNA for the same cell line. No-amplification blanks were used as negative controls. DNA fragment analysis was then conducted on an Applied Biosystems 3730, and the data were analyzed using GeneMarker software (SoftGenetics, State College, PA, USA).

Genomic DNA was amplified by polymerase chain reactions carried out in a total volume of 10 µL, the solution contained 5.40 µL of H₂O, 2.0 µL of green buffer, 0.8 µL of MgCl₂, 0.125 µL dNTP, 0.25 µL of primer forward, 0.30 µL of primer reverse, 0.04 of Taq-polymerase and 1 µL (5 ng) of DNA extract. The Eppendorf thermocycler was programed with an initial denaturing cycle at 95 °C for 2 min, followed by 45 cycles of a denaturing step at 95 °C for 30 s, followed by an annealing step (temperature varied depending on the microsatellite; Table 1) for a period of 30 s, and a final elongation step at 72 °C for 40 s.
Allele sizes were estimated using a DNA fragment analyzer (ABI 3730xl DNA). We used ABI DS-33 dye set with G5 filter set. Forward primers were dye labeled with either 6-FAM, VIC, NED or PET dye labels; GS-600 standard set was used with LIZ dye. This protocol allowed the detection of several PCR products at the same time by fluorescence emission. Fragment sizes were estimated using GeneMarker^® software (SoftGenetics, State College, PA, USA).

The targeted copy number of Ikaros family zinc finger 1 (IKZF1) was analyzed using a SALSA MLPA Probemix P335-C1 ALL-IKZF1 kit (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The MLPA results were analyzed using GeneMarker software (Softgenetics, State College, PA, USA). Peak heights were normalized, and deletions or duplications were defined as recommended by the manufacturer. Direct sequencing of the probe-binding and ligation sites was performed for the relevant exons to detect nearby variants, which can lead to a false decrease in peak signal [24 (link)].

For genotyping of the cell lines the GenomeLab STR Primer Set Kit (Beckman Coulter, Krefeld, Germany) and the Amplitaq GOLD DNA Polymerase (Life Technologies, Carlsbad, USA) were used, respectively. The analysis was performed according to the manufacturer’s handbook. PCR products were separated on the CEQ GeXP capillary electrophoresis system (Beckman Coulter). 12 alleles were determined using the Gene marker software (Softgenetics, State College, USA).