Supplemental Digital Content 1
Supplemental Methods
Plasmid construction
The pcDNA3.1(+)-v1 and pcDNA3.1(+)-v3b fusion plasmids were constructed as described previously 12. The primer sequences used for plasmid construction are shown in Supplemental Digital Content 2 (Table). Total cDNA from H2228 cells was used as the template for construction of pCR2.1-alk. The PCR products were cloned by TA cloning (Invitrogen). For the construction of pcDNA3.1(+)-v2, -v3a, -v4, -v5a, -v5b, -v6, and -v7, two or three PCR fragments were inserted into EcoRV (New England Biolabs, Beverly, MA)–digested pcDNA3.1(+) (Invitrogen) by In-Fusion PCR cloning (Clontech). Total cDNA from H2228 cells was used as the template for ALK and EML4. The additional insertion of the ALK gene in variant 5b was generated with genomic DNA from H3122 cells. Synthetic oligonucleotides were used for the unknown sequence in variant 6. The other plasmids contained ~1000 to 2000 nucleotides, including each fusion point between EML4 and ALK.
Detection of EML4-ALK
Both PCR and the single-base primer extension reaction (Figure 1) were performed with the use of iPLEX chemistry in a MALDI-TOF mass spectrometer (Sequenom). PCR primer pairs were designed such that the forward and reverse primers were targeted to EML4 and ALK, respectively. Each single-base extension primer was designed with the MassARRAY Assay Design software (version 4.0, Sequenom) to hybridize across the fusion point, spanning 2 to 10 bases on the EML4 side of the fusion gene. The sequences of the PCR and single-base extension primers are provided in Supplemental Digital Content 3 (Table). The RT-PCR reactions were performed in a total volume of 5 µL in standard 96-well plates. The reverse-transcribed cDNA was subjected to PCR with 1 U of Taq polymerase, 500 µmol of each deoxynucleoside triphosphate, and 200 nmol of each PCR primer. The thermal cycling was performed in an ABI-9700 instrument (Applied Biosystems) for 2 min at 95°C followed by 45 cycles of 30 s at 95°C, 30 s at 56°C, and 60 s at 72°C and then a final incubation for 5 min at 72°C. After completion of PCR, 2 µL (0.5 U) of shrimp alkaline phosphatase (Sequenom) were added to each reaction and the resulting mixture was incubated for 40 min at 37°C before inactivation of the enzyme by incubation for 5 min at 85°C. The single-base primer extension reaction was then performed in a final volume of 9 µL containing 1 µmol of each extension primer, 0.2 µL of mass-modified deoxynucleoside triphosphates (Sequenom), and 0.04 µL of iPLEX enzyme (Sequenom). The thermal cycling program for the reaction included an initial denaturation for 30 s at 94°C followed by five cycles of 5 s at 52°C and 5 s at 80°C. An additional 40 annealing and extension cycles were then looped back to 5 s at 94°C, 5 s at 52°C, and 5 s at 80°C. The final extension was performed at 72°C for 3 min, and the samples were then cooled to 4°C. The reaction products were desalted by dilution with 41 µL of distilled water, the addition of 15 mg of resin (Sequenom), and subsequent separation of the resin by centrifugation. The products were spotted on a SpectroChip (Sequenom), processed, and analyzed with a Compact Mass Spectrometer and MassARRAY Workstation (version 3.3) software (Sequenom). Data analysis was performed with MassARRAY Typer software version 4.0 (Sequenom).
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