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
1
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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