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Supplementary Material and Methods
Long distance inverse PCR
The tumor DNA was digested at 37°C for 2 hours with the restriction enzymes SphI, MfeI,
XbaI (Fermentas, St. Leon-Rot, Germany) followed by heat-inactivation at 68°C for 30
minutes. To circularize the resulting DNA fragments, 40 ng of the digested DNA were ligated
at 4°C overnight in a total volume of 50 µL with 2 U of T4 DNA ligase (Invitrogen,
Groningen, Netherlands). The ligated DNA was then isopropanol precipitated and dissolved
in a final volume of 10 µl Tris. The oligonucleotides in the first round of amplification were:
MfeI
ex:
5’-GCTCAGCTAAGAAATACTAGCACAGTG-3’,
SphI
ex:
5’-
CTTTAGAATTATGAGGTGCGCTGTGTG-3’,
XbaI
ex:
5’-
CCTTGTCTAAGTCATTGACTGTAGGTC-3’,
JH6
ex:
5’-
TGCAATGCTCAGGAAACCCCACAGG-3’. PCR reaction mixture (30 µL total volume)
contained 9 µL of 3.3x Gene Amp XL Buffer II (Applied Biosystems, Foster City, CA, USA),
200 µmol/L dNTPs, each primer at 0.2 µmol/L, 0.4 U rTth polymerase (Applied Biosystems),
1.25 mmol/L Mg(OAc)2 and 10 µL of the ligated DNA. Cycling conditions for the first round
of PCR were 4 min at 95°C, 34 cycles of 45 s at 95°C, 45 s at 66°C and 8 min at 68°C,
followed by a final extension step of 15 min at 72°C. Nested amplification with the internal
primers (MfeI int: 5’-GCTGGAAGCAGATGATGAATTAGAGTC-3’, SphI int: 5’AGAGAACTGTCGGAGTGGGTGAATC-3’,
XbaI
int:
5’-
ACCCTTGAAAGTAGCCCATGCCTTC-3’,
JH6
int:
5’-
TAGCAGAAAACAAAGGCCCTAGAGTG-3’) was performed for 45 cycles using identical
conditions. The amplificates were purified by agarose gel electrophoresis following standard
procedures and bidirectionally sequenced with the oligonucleotides used for amplification,
using the BigDye kit (Applied Biosystems) and an automated sequencing apparatus
(ABI3100, Applied Biosystems). Sequences were compared to published sequences using the
BLAST tool (www.ncbi.nlm.nih.gov/BLAST). The presence of the chromosomal
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translocations amplified from NHL in microdissected HRS cells was verified using the
following primers in semi-nested PCRs together with the JH6 int primer: MCRext 5´and
CCCCATTGAGGTAGGTGATTATGT-3´
TTTGGTTTCATGCGAACTAACAAAG-3
for
GGCCACAGTCCTATCAGACTAGAGC-3´
case
1,
and
ATCCAGATGGCAAATGACCAGCAG-3´
ATCTATGGTGGTTTGACCTTTAGAG-3´
MCRint
for
case
2
and
and
MBRext
5´5´-
MBRint
5´-
CoSWCHR11.1
5´-
CoSW11.2
5´-
GATGACCAACCACCTGTCCAGACC-3´ for case 5. All reactions were carried out in a two
rounded PCR with the following cycling conditions: 180 s at 95°C, 35 cycles for the first or
45 cycles for the second round of PCR consisting of 30 s at 95°C, 60 s at 61°C, 45 s at 72°C
and a final extension of 180 s at 72°C.
Analysis of allele-specific expression of BCL2 in FL/HL composite lymphomas
To screen for monoallelic single nucleotide polymorphisms (SNPs), which would allow us to
discriminate the translocated and the non-translocated BCL2 alleles in cases 1 and 2 we first
sequenced the coding regions of the BCL2 gene in cases 1 and 2 using genomic DNA from
whole tissue sections. In case 1 we found one SNP whereas no SNP was detected in case 2.
We designed specific primers to detect the SNP in case 1 in a semi-nested RT-PCR (BCL2798-Pol-US 5´-CAGGATATTTAATGACAACCTTCTGG-3´, BCL2-798-Pol-LS1 5´GAATGATTCACTGGGTAAGACTAAAGG-3´, BCL2-798-Pol-LS2 5´ACTCTCCAATTCAGTTTCACATTGC-3´). Since the breakpoint in this case is located
approx. 30 kb downstream of the BCL2 coding sequence it was not possible to amplify the
breakpoint and polymorphic position in a single PCR product.
RNA was extracted from 300 laser microdissected tumor cells of each FL and HL
using the Purescript kit following the recommendations of the manufacturer (Gentra,
Minneapolis, MM, USA). Each sample was divided into two parts and a cDNA synthesis was
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performed in parallel with a control reaction without addition of reverse transcriptase (-RT
control) using a Roche 1st strand cDNA synthesis kit (Roche, Mannheim, Germany).
cDNA was diluted to single molecule level and a PCR with the SNP-specific primers
was performed. –RT control reactions were used as negative control, which were negative for
all 16 analyzed FL samples. However, in 2 of 24 negative controls from the HL samples the
product from the translocated allele was amplified. Because the PCR was not intron spanning,
we assume that these products are DNA contamination deriving from incomplete DNAse
digestion. Cycling conditions for the first round of PCR were 180 s at 95°C, 35 cycles of 30 s
at 95°C, 60 s at 61°C and 45 s at 72°C, followed by a final extension step of 180 s at 72°C. In
the second round of PCR 45 instead of 35 cycles were performed. All other conditions
remained unchanged. The amplicons were purified by agarose gel electrophoresis and
bidirectionally sequenced with the oligonucleotides used for amplification.
Analysis of the ATM gene
Mutation analysis of the ATM gene was performed by denaturing high performance liquid
chromatography (dHPLC) using genomic DNA of the corresponding NHLs. Briefly, PCR was
carried out for each of 62 coding exons and flanking intronic sequences using 60 primer pairs
modified from a previous protocol.(1) Reactions contained 50 ng genomic DNA, 0.2 µmol/L
dNTPs, 0.2 µmol/L each primer, 1.5 mmol/L MgSO4, 1 U Optimase (Transgenomic, Omaha,
NE, USA). Cycling conditions were 5 minutes at 95°C, followed by 40 cycles of 30 s at 95°C,
30 s at 53°-58˚C and 60 s at 72°C, and finally 7 minutes at 72°C. PCR products were heated
to 95°C for 5 minutes then cooled slowly to 65°C, to allow formation of any potential
heteroduplexes. Reaction products were analysed by dHPLC using the WAVE® DNA
fragment analysis system (Transgenomic). The temperature for analysis for each exon was
predicted using Wavemaker software (Transgenomic). Chromatograms were analysed
visually and the PCR product was sequenced if a variant pattern was detected.
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Mutational analysis of the tumor suppressor genes TP53, NFKBIA and FAS
For single or few cell PCR analysis samples were incubated with 0.5 mg/mL proteinase K
(Roche) for 4 h at 55°C and then 10 min at 96°C. Exons 3-6 of the NFKBIA gene were
amplified by PCR from genomic DNA as described.(2) Exons 1 and 2 of NFKBIA were
amplified
in
the
first
round
of
PCR
using
the
oligonucleotides:
1a:
5’-
GCCGACGACCCCAATTCAAAT C-3’, 1c: 5’-CATCGCTGGTCCCCCGGCTC-3’, 2d: 5’GCTACGTCCCAGGGTCAGAGAG-3’, 2a2: 5’-TGGTGGATGGCAGCGTCGCCACAC3’. Semi-nested amplification of each exon was performed using 1a, 1r: 5’GCGTCCCGCCCTCCCGACGA-3’, 2d, 2b2: 5’-GCCTGCCAGGAACACTCAGCTC-3’.
PCR conditions were as described,(2) with the modification that the reactions were
supplemented with 1.5x PCR Enhancer (Invitrogen). Exon 9 of the FAS gene was amplified
from genomic DNA according to a published protocol.(3) Exons 7 and 8 of the FAS gene
were amplified using the oligonucleotides: E7F: 5’-CTTCTTATATTTCTCTTAGTGTG-3’,
E7R:
5’-CTCCATCTCAAACAAAATGAAAC-3’,
TTAAGGAAAAATTAGAAGTTCACA-3’,
E8F:
E8R:
5’5’-
GTAGAATGTATGAGAAATAACAAG-3’ in the first round of amplification. Reaction
mixes were supplemented with 0.125 µmol/L of each primer and 3 mmol/L MgCl 2 for exon 8
and 2 mmol/L MgCl2 for exons 7 and 9. After incubation for 5 min at 95°C, 35 cycles of 50 s
at 95°C, 30 s at 55°C, and 60 s at 72°C were followed by 10 min at 72°C. Nested
amplification of the individual exons was performed using identical conditions with internal
primers
(E7Fi:
5’-TATGTTCTCACATGCATTCTAC-3’,
AAGTAACAAAAAGCCAAATCAC-3’,
E8Fi:
E7Ri:
5’-
5’-TTTATTTGTCTTTCTCTGCTTC-3’,
E8Ri: 5’-CCATAATATGTCACTGAAATTAG-3’).
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References for supplementary Material and Methods
1.
2.
3.
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Hansmann ML, Rajewsky K, Küppers R. Clonal deleterious mutations in the
IkappaBalpha gene in the malignant cells in Hodgkin's lymphoma. J Exp Med
2000;191:395-402.
Müschen M, Re D, Bräuninger A, Wolf J, Hansmann ML, Diehl V, Küppers R,
Rajewsky K. Somatic mutations of the CD95 gene in Hodgkin and Reed-Sternberg
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