Supplementary Method 3. Array-MAPH hybridizations.
(a) Hybridization and washing of filters
Filters were prehybridized at 65°C overnight in 1 ml prehybridization solution containing (0.5M
Na2HPO4/NaH2PO4 (pH=7.2); 7% SDS; 1mM EDTA, 100 µg/ml denatured herring sperm
DNA). Then the prehybridization solution was replaced by 200 µl prehybridization solution;
containing 2 µg denatured human Cot-1 DNA (Roche, Basel, Switzerland) and filters were
incubated at 65°C for 1 hour. A mixture of all probes (2 ng each per filter), together with 1µg per
filter of human Cot-1 DNA (Roche) and 20 pmol of each PZA/PZB and UNI3/UNI5 blocking
mixture were denatured with 4.5µl per filter of 1 M NaOH at 37°C for 1 min. Denatured probe
mix was placed onto ice, neutralized with 7 µl of 1 M Na2HPO4/NaH2PO4 (pH=7.2) per filter
and added to hybridization mixture. Filters were hybridized at 65°C overnight. Hybridization
solution was removed from filters, replaced with 1 ml prehybridization solution followed by
washes in wash solution 1 (1xSSC; 1% SDS) and wash solution 2 (0.1xSSC; 0.1% SDS)
prewarmed to 65°C.
(b) Recovering of bound probes by PCR amplification
Each filter was placed into a separate tube, bound probes were recovered by denaturing from
filters and amplified for five cycles at 94°C for 1 min, annealing and extension at 70°C for 1 min
in 50 µl reactions containing 15 pmoles PZA and PZB universal primers, 1mM MgCl 2, 0.2mM
each dNTP and 2.5U Taq DNA Polymerase. A secondary amplification was carried out from 2
µl of the primary amplification product in 20 µl with 70 pmoles of PZA and PZB primers, 2mM
MgCl2 concentration, 0.2mM of each dNTP, and 1U Taq DNA Polymerase, for 20 cycles at
94°C for 1 min denaturation, followed by annealing and extension at 70°C for 1 min with final
extension for 20 min at 70°C.
(c) Labeling of recovered probes and preparation for hybridization.
The amplified probes were treated with shrimp alkaline phosphatase (SAP) and exonuclease I.
To 20 µl of amplified MAPH probe mixture, 1.5 U of exonuclease I (MBI Fermentas, Vilnius,
Lithuania) and 1.35 U of SAP (USB Corporation, Cleveland, OH, USA) were added and
incubated for 30 min at 37°C. Reactions were stopped by heating for 15 min 80°C. Probes were
purified using a PCR product cleaning kit (MoBio Laboratories, Solana Beach, CA, USA)
according to manufacturer protocol and dried down by vacuum concentration. Probes were then
dissolved to 38.5µl in ddH2O and labeled by nick translation using aminoallyl-dUTP allowing
for later treatment with amino-reactive Cy3 or Cy5 dyes. The nick translation reaction was
mixed on ice, in a volume of 50 μl, with 20μM dATP, 20μM dCTP, 20μM dGTP, 50mM Tris
HCl (pH=7.8), 5mM MgCl2, 10mM mercaptoethanol, 0.02mM 5-(3 aminoallyl)-2′-deoxyuridine5′-triphosphate (aa-dUTP) (Sigma-Aldrich, St. Louis, MO, USA), 0.05μg BSA (Promega,
Madison, WI, USA) and 5μl DNA Pol I (0.5U/µl)/ DNAse(0.4mU/µl) mix (Invitrogen Co,
Carlsbad, CA, USA,). Reaction was incubated at 15C for 90 minutes, stopped by incubating at
80C for 15 minutes and kept at room temperature for 20 min. For purification, PCR purification
kit (MoBio Laboratories) was used. Dehydration was performed in vacuum concentrator. The
probes were resuspended in 4.5 μl of 0.1M Na2CO3 (pH=9.0) (made freshly, heated to 60C for
15 min to inactivate DNase) and 4.5 μl Cy3 monoreactive-dye (GE Healthcare UK Ltd, Little
Chalfont Buckinghamshire, UK), one tube dissolved in 45µl of 100% DMSO, was added.
Samples were incubated in a dark place at room temperature for 1 hour. 3.5μl of 4M
hydroxylamine (Sigma-Aldrich) was added and samples were incubated in a dark place at room
temperature for 15 minutes. 35 μl of 100mM sodium acetate was added and probes were purified
with PCR purification kit (MoBio Laboratories) and dehydrated in vacuum concentrator.
(d) Microarray preparation and hybridization of the recovered probes.
Fluorescently-labeled probes were dissolved in 80 µl of degassed hybridization buffer (50%
formamide, 6x SSC, 0.5% SDS and 5x Denhardt’s solution). Following solutions were prepared
and degassed for 5 minutes: pre-wash solution: 6X SSC, 0,5% SDS; solution 1: 2X SSC, 0.03%
SDS; solution 2: 1X SSC; solution 3: 0.2X SSC. Hybridizations were performed using
automated hybridization station HS-400 (Tecan Austria GmbH, Grödig/Salzburg, Austria) using
following program:
1 Wash the glasses using pre-wash solution for 1 minute at 85°C, soak time 30 sec.
2 Probe injection at 60°C
3 Denaturation at 95°C for 5 minutes
4 Hybridization at 42°C for 8 hours, medium agitation mode
5 Wash at 42°C using solution 1, 1.5 minutes, 30 s soak time, 3 cycles
6 Wash at 42°C using solution 2, 1.5 minutes, 30 s soak time, 3 cycles
7 Wash at 42°C using solution 3, 1.5 minutes, 30 s soak time, 3 cycles
8 Dry slides with N2, 1.5 minutes at 30°C