Supplementary Methods
DNA Extraction and Quantification
DNA was extracted from tumors received either freshly frozen or formalin-fixed and
paraffin embedded (FFPE). Many of the DNA extractions were completed by collaborators from
other laboratories and their methods of extraction differed. DNA was extracted by RecoverAll
Total Nucleic Acid Isolation Kit for FFPE (LifeTechnologies), DNeasy Blood and Tissue Kit
(Qiagen) or using a phenol/chloroform method.
Extraction by the phenol/chloroform method was done as follows. DNA was extracted
from pellets containing ~1 million cells or more, using TE pH 8 with 0.5% SDS buffer and 250
μg/mL Proteinase K. DNA was purified using phenol and phenol-chloroform isoamyl alcohol
(25:24:1) buffers, and then precipitated in 95% ethanol at -20 °C overnight. Following
precipitation, DNA extract was centrifuged at 12000 RPM for 30 minutes and washed with 70%
ethanol. DNA was air dried and dissolved in Tris-HCl. Concentration was measured by
spectrophotometry (Nano-drop 8000, Thermo Scientific).
Extraction by individual commercial kits was done according to the protocols of each
individual kit and company.
Terminal Restriction Fragment (TRF)
Alternative lengthening of telomeres (ALT) was detected using TRF. TRF is a southern
blot based assay which allows for the visualization of overall range of telomere length.
Telomeric DNA is separated based on size and the telomere spread is blotted on a membrane.
An ALT positive tumor has very long telomeres and a heterogeneous karyotype.
For each sample, 2 µg of DNA was digested with 10 U/µg of both HinfI and RSAI
restriction endonucleases, for 2 hours at 37 °C. Following digestion, samples were loaded onto a
14 cm 0.8% agarose gel and electrophoresed at 60-75 V until loading dye had run ~3/4 (10-11
cm) of the length of the gel. Southern blotting was done onto a 2x SSC soaked Hybond-N+
(Amershand) membrane. Samples were crosslinked at 1200 J. Prehybridization and
hybridization were done using the TeloTAGGG kit from Roche.
Telomere Specific Fluorescent In Situ Hybridization (FISH)
ALT was detected by FISH on paraffin slides. An ALT positive FISH was one where
there were large fluorescent foci, with a large range in focus size between large and small foci, as
compared to negative controls.
Each paraffin slide was de-waxed in xylene for 30 minutes after which the sample was
placed in 100% ethanol. They were further treated with RNase diluted in 2x SSC and incubated
for 1 hour at 37 °C and later washed twice with 2x SSC. The slides were incubated at 80 °C for 8
minutes in 1 M NaSCN. Slides were rinsed twice with water, and were incubated in 5 mg/mL
pepsin in 0.9% NaCl pH 1.56 for 7-9 minutes at 45 °C. Slides were rinsed twice in 2x SSC for 1
minute and placed in a solution of 0.1M triethanolamine pH 8.0 with 0.25% v/v acetic acid for
acetylation for 10 minutes. Following acetylation, slides were rinsed in PBS twice, for five
minutes each, and were then soaked in 2x SSC for 5 minutes. Finally, the slides were dehydrated
through ethanol series washes of 70%, 90%, and 100% for 2-3 minutes each.
The probe was diluted in probe solution constituting (70% formamide, 5% MgCl2, 10mM
Tris-HCl, 1% Blocking Reagent) The probe (PNA Bio, U.S.A) was added to the tissue and
sealed with rubber cement. The slide was denatured at 80 °C for 3 minutes and placed overnight
at 37 °C overnight. Following hybridization, the coverslip was removed in wash solution I (70%
formamide, 10 mM Tris, 0.1% BSA pH 7.0-7.5). The slide was then washed twice, for 15
minutes each wash, in wash solution I, 3 times, for 5 minutes each, in wash solution II (0.1 M
Tris, 0.15M NaCl, 0.08% Tween pH 7.0-7.5). Slides were then dehydrated through ethanol
series. 0.1 µg/mL of DAPI/antifade was placed on the coverslip, and was then covered with the
slide.
C-circles Assay (CCA)
ALT was detected by screening for c-circles. C-circles are extrachromosomal telomeric
repeats (ECTRs) which are ALT specific. They have been found to be robustly associated with
ALT and are present in tumors which maintain their telomeres using a recombination phenotype,
even following loss of other ALT phenotypic markers [1]. The c-circle assay was performed
using two different detection methods. The first was detection of c-circles by dot blot (southern
blot) and the second was detection using a telomere-specific qPCR assay.
Detection of C-circles using dot blot
DNA was diluted to 25 ng/μL. 100 ng of DNA was digested using 10 U/μg of HinfI
(New England Biolabs - R0155) and RsaI (New England Biolabs - R0167) for 2 hours at 37°C
(final volume of 7 μL). DNA was then treated with 12.5 U/μg Lambda exonuclease (New
England Biolabs – M0262) for 45 minutes at 37 °C (final volume of 10 μL), then 30 U/μg of
exonuclease V (New England Biolabs – M0345) for 45 minutes at 37 °C (final volume of 13 μL)
and finally 100 U/μg of exonuclease I (New England Biolabs – M0293) for 50 minutes at 37 °C
(final volume of 17 μL), all according to the manufacturer’s instructions.
Half the total volume (8.5 μL, or 50 ng) was transferred to a clean PCR tube. Volume
was topped up to 10 μL. To this 10 μL volume was added 10 μL of phi29 master mix
containing: 4mM DTT, 10x Φ29 buffer, 0.2 mg/mL BSA, 0.1% Tween, 25 mM (each) of dATP,
dGTP, dTTP, and dCTP, 15 U of Φ29 polymerase.
The sample mixture was incubated for 8 hours at 30 °C followed by an inactivation stage
of 20 minutes at 65 °C. 40 μL of 2x SSC was added to the sample mixture and then it was
denatured at 100 °C for 10 minutes, and then kept on ice.
The samples were blotted onto a 2x SSC soaked Hybond-N+ (Amershand) membrane
using the BioDot apparatus (BioRad). Samples were crosslinked twice at 1200 J.
Prehybridization and hybridization were done using the TeloTAGGG kit from Roche.
Detection of C-circles using qPCR
DNA was diluted to 3.2 ng/μL. 16 ng (5 μL) of DNA was treated with 5 μL of Φ29
master mix containing: 4 mM DTT, 10x Φ29 buffer, 10 μg/μL BSA, 0.1% Tween, 1 mM (each)
of dATP, dGTP, dTTP and dCTP, 5 U of Φ29 polymerase.
Following the Φ29 amplification, the samples were diluted to 0.4 ng/µL by adding 30 μL
of sterile water to the reaction mixture. Un-treated dilutions of 0.4 ng/μL were also prepared.
The qPCR assay was run with triplicates of Φ29 treated and untreated DNA, requiring a
total of 6 PCRs per sample. Each qPCR required 15 μL of master mix and 5 μL (2 ng) of DNA.
The qPCR master mix contained: 1x QuantiTECT SYBR Green Master Mix (LifeTechnologies
- 4309155), 10 mM DTT, 0.5 μL DMSO, and 300 nM of forward primer and 400 nM of reverse
primers. All qPCRs were done in 96 well plates using the Lightcycler 480 (Roche). PCR
conditions were: 95 °C for 15 minutes, 35 cycles of 95 °C for 15 seconds (denaturation), and
54 °C for 2 minutes (annealing and elongation). Lightcycler 480 software was used to obtain
data.
For determining presence of c-circles, a ΔmeanCp value was calculated for each sample.
The ΔmeanCp was the difference between the mean values of the non-Φ29 and Φ29 treated
samples. A positive ΔmeanCp value greater than 0.2 was detected in c-circle positive samples,
whereas a negative ΔmeanCp value of less than -0.2 was detected in c-circle negative samples.
Samples with ΔmeanCp -0.2 – 0.2 were considered as atypical, and results were confirmed using
the dot blot.
ATRX and p53 Immunohistochemistry
ATRX immunohistochemistry was done using the Benchmark XT (Ventana, Cat. # 750700) platform with optiview DAB IHC detection kit (Ventana, Cat. # 760-700). ATRX
polyclonal antibody (Sigma Aldrich, Cat. # HPA001906) was used at a 1/400 concentration. p53
immunohistochemistry was done as previously described[2] using a monoclonal antibody
concentration of 1/250.
1.
Henson JD, Cao Y, Huschtscha LI, Chang AC, Au AYM, Pickett HA, Reddel RR (2009)
DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity.
Nat Biotechnol 27:1181-1186
2.
Li L, Mu K, Zhou G, Lan L, Auer G, Zetterberg A (2008) Genomic instability and
proliferative activity as risk factors for distant metastases in breast cancer. Br J Cancer 99:513-519