DNA Fluorescence in situ hybridisation (FISH)

Protocol Title: DNA Fluorescence in situ hybridisation (FISH)

Karin Purshouse¹, Wendy Bickmore¹

1 - MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, EH 4 2XU

Abstract: 

DNA FISH is a technique that allows direct visualisation of genomic loci. This protocol has been previously published (Jubb and Boyle, 2020), and optimised here for the visualisation of ecDNA in primary glioblastoma stem cells (Purshouse et al., 2022). 

Materials and reagents:

Equipment:

• 37°C shaking incubator for overnight culture
• Fume hood
• Centrifuge
• Water baths (see method for temperatures) with metal trays
• Bench materials outlined in methods

Procedure:

Fosmid probe preparation

Fosmid probes were ordered from the WIBR2 Library (BACPAC Genomics Inc, Emeryville, California).  A bacterial stab from the probe was streaked onto a plate of LB-Agar with 12.5ng/μL chloramphenicol and incubated overnight (o/n) at 37°C.  A single colony was picked and cultured o/n at 37°C in a shaking incubator, in a 50ml Falcon tube containing 5ml L-broth with 12.5ng/μL chloramphenicol, including a stab-free culture tube as a negative control.  A glycerol stock for each fosmid was prepared using 800μl of the o/n culture suspension with 400μl 100% glycerol, which was stored long term at -80°C.

An alkaline miniprep was performed to generate fosmid DNA. An o/n culture of bacteria containing the relevant fosmid probe was centrifuged (5 mins at 4000 x g), the supernatant discarded and the pellet resuspended vigorously in 200μl GTE buffer (50 mM Glucose, 25 mM Tris pH 8, 10 mM EDTA) with 50mg lysozyme.  After incubating for 5 mins at room temperature (r.t), 400 μL lysis buffer (0.2 M NaOH, 1% SDS) was added, the solution mixed by inversion and incubated on ice for 5 mins.  Then 300 μL acetate buffer (3M potassium acetate, 11.5% glacial acetic acid prepared in dH2O) was added and mixed gently but thoroughly by vortexing to ensure flocculation could be observed. After incubating on ice for 5 mins, samples were centrifuged (16,000 x g, 5 mins, 4°C) and the supernatant transferred to a fresh 1.5ml Eppendorf tube.  DNA was purified by phenol:chloroform extraction, 500μL phenol:chloroform were added, mixed by inversion and centrifuged (16,000 g, 4 mins, 4°C). The top layer was transferred to a fresh tube and this process was repeated with chloroform. DNA was precipitated from the aqueous phase with 500μl isopropanol. After >1 h at -20°C DNA was precipitated by centrifugation (16,000 g, 15 mins, 4°C). The pellet was washed in 70% EtOH and dried before resuspension in 25μl Tris/EDTA pH 8.0 (TE) supplemented with 2µl 20mg/ml RNase A (Invitrogen). This was incubated for 5 mins at 37°C before long term storage at -20°C.  Fosmid DNA was quantified with the Qubit dsDNA broad range assay (Thermo Fisher) using the Qubit 4 fluorometer.  

All fosmids were validated to ensure they bound specifically and sensitively to the intended locus prior to use. Validation was either by performing DNA FISH on an existing Me:ac preparation of Neo3 cells, a human parental lymphoblastoid cell line with a normal karyotype, or by performing FISH on NS9 cells with a previously validated fosmid binding to the same chromosome. 

Nick Translation

Fosmid DNAs (6μl - (0.5 – 1 μg)) were directly labelled by nick translation to incorporate a fluorescent dUTP (2.5μl) (Green496-dUTP, ENZO life sciences; ChromaTide AlexaFluor 594-5-dUTP, Thermo Fisher Scientific) by incubation with 2.5μl each of unlabelled 0.5mM dATP, dCTP and dGTP, 1ul ice cold DNase I (Roche; 1:5 dilution in ice cold water), 2 μL nick translation salts (0.5 M Tris pH 7.5, 0.1 M MgSO₄, 1 mM dithiothreitol (DTT) and 0.5 mg/ml BSA) and 1μl DNA polymerase I (Invitrogen) for 90 min at 16°C. The reaction was quenched with 3μl 0.5M EDTA and 2μl 20% SDS, TE buffer added to a total volume of 90μl and the reaction mix purified using a Quick Spin Sephadex G50 column (Roche). Once labelled, fosmid probes were stored at -20°C. 

Probe Hybridisation

Cells on slides or cover-slips for 3D FISH were prepared by briefly rinsing them in 2x Trisodium citrate and sodium chloride (SSC).  Cells on slides for 2D FISH (metaphase spreads dropped in Me:Ac fixative) were additionally dehydrated for 1 h at 70°C if they had been dropped the same day.  

Slides were incubated for 1 h in 2xSSC with 100 μg/ml RNaseA (Invitrogen) at 37°C in Coplin jars, then dehydrated in 70%, 90% and 100% EtOH for 2 minutes each.  After air drying, slides were incubated for 5 minutes at 70°C and immersed in a denaturing solution (2xSSC/70% formamide, pH 7.5) heated to 70°C (Me:ac-fixed cells) or 80°C (pFA-fixed cells) for 1-2 mins for 2D DNA FISH, depending on the timeframe since the cells were dropped, and 40 mins for 3D DNA FISH. Slides were immediately immersed in ice-cold 70% ethanol, followed by 90% and 100% EtOH at r.t., for 2 mins each before air drying. 

The only deviation from this protocol was for slides where RNA FISH had previously been performed on a slide.  Here, and following imaging of RNA foci, slides were transferred directly from PBS into a denaturing solution at 80°C for 15-30 mins without the preceding 2xSSC or EtOH washes.  Following denaturing, slides were immediately washed in 2xSSC and minimally dried.  

FISH probes were prepared by combining approximately 80-100ng of each directly labelled fosmid probe (per slide), 6 μg Human Cot-1 DNA (per probe; Invitrogen), 5 μg sonicated salmon sperm (per slide; Invitrogen) plus two volumes of 100% ethanol. The probe mix was pelleted and dried.  The pellet was suspended in hybridisation mix (50% deionised formamide (DF), 2× SSC, 10% dextran sulphate (Sigma), 1% Tween 20 in ddH2O) by vortexing and briefly centrifuging, and then incubated for 1 h at r.t. in the dark.  Alternatively, in order to perform chromosome territory analysis, FISH probes were instead suspended in 10μl of Chromosome 7 paint (XCP 7 Orange, Metasystems) and incubated in the same way. Probes were then denatured for 5 mins at >70°C and annealed at 37°C for 15 min. Probes were then hybridised to the denatured slides under a sealed coverslip o/n at 37°C.  

The following day, slides were washed four times for 3 mins each in 2xSSC (45°C) then 4x 3 mins in 0.1% SSC (60°C), then immersed in 4xSSC/0.1% Tween 20 with 50ng/ml DAPI for 3 mins. Slides were mounted with 25-50μl of Vectashield onto an appropriately sized coverslip and sealed with clear nail varnish. 

Where noted, an additional Centromere 7 (CEN7 - CHR07-Dig Control) FISH probe (Pisces Scientific) was prepared by combining 2μl CEN7 probe with 8μl of CEN7 hybridisation mix.  This was denatured for 5 min at 80°C and snap frozen on crushed ice. This CEN7 hybridisation mix was combined with the relevant DNA FISH probe/hybridisation mix which was prepared as above except in a slightly greater volume (20μl).  This was applied directly to denatured slides and hybridised overnight as above. Following washing (see above), slides were incubated at 37°C in a humidified chamber with 50μl blocking buffer (4x SSC/5% Marvel) for 5 mins.  This was followed by a 1 h incubation with 50μl anti-digoxigenin antibody (Roche; 1 in 10) and a 1 h incubation with 50μl anti-sheep Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific; 1 in 10) in the same conditions with 4xSSC/0.1% Tween 20 washes (3 x 2 mins) in between. After the final wash, slides were stained with DAPI and mounted with Vectashield as above.

Immuno-FISH

For immuno-FISH (DNA), IF was performed as outlined elsewhere.  Following the final washes after secondary antibody incubation, slides were incubated with 4% pFA for 30 mins at r.t. to fix the signal. Following thorough PBS washes (three times), the DNA FISH protocol was then followed as above.

For immuno-FISH (RNA), the antibodies were added at the same concentration as described above directly to the hybridisation mix (primary antibody) and 2xSSC/10% DF washes (secondary antibody), without further deviation from the RNA-FISH protocol outlined in Purshouse et al, 2022. 

References:

References:

Jubb A and Boyle S (2020) Visualizing genome reorganization using 3D DNA FISH In: Nielsen BS, Jones J, editors. In Situ Hybridization Protocols. New York, NY: Springer. pp. 85–95.

Purshouse K, Friman ET, Boyle S, Dewari PS, Grant V, Hamdan A, Morrison GM, Brennan PM, Beentjes SV, Pollard SM, Bickmore WA (2022) Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells eLife 11:e80207

These methods were extracted from the following PhD thesis: https://era.ed.ac.uk/handle/1842/40874