Dot blot hybridisation

2.4.1 Preparation of dot blots

After gel analysis, PCR reaction tubes were placed in a PX2 Thermal Cycler (Thermo Electron Corporation), heated to 95°C for 10 minutes, and quickly chilled on ice. An aliquot (1 μL) of each PCR product was spotted onto a pre-marked membrane

(Hybond N+, Amersham) and allowed to dry. The DNA was fixed to the membrane by UV cross linking, by placing the membrane wrapped in a saran wrap, DNA side up, in a UV cross linker for 5 minutes at 2000 μJ/cm2. The blots were stored dry at 4°C until use. The PCR reactions for EAdV were not evaluated by dot bot hybridisation.

2.4.2 Preparation of probes

Probes were prepared using the DIG (Dioxigenin) probe synthesis kit. Virus specific DIG-labelled DNA probes were generated by incorporation of DIG into PCR products during amplification. The probes were prepared using a PCR reaction mix consisting of a 5 μL of 10x PCR buffer with MgCl2, 5 μL of 10x PCR DIG mix, 1 μL of 20 μM primers (forward and reverse, see table 1), a final concentration of 2.6.5 U of Expand high fidelity enzyme mix and sterile re-purified water in a total volume of 49 μL. Control reactions were prepared in parallel, consisting of the same PCR reaction mix, with 5 μL 10x dNTP stock solution instead of PCR DIG mix. Template DNA (1 μL) was then added to the probe mix and to the DIG-control mix, and amplified in the PX2 Thermal Cycler (section 2.5.3). The results were analysed by gel electrophoresis (section 2.5.5). The product size of the template probe was compared to the probe control to confirm the incorporation of DIG in the probe template DNA.

2.4.3 Sequencing of probe controls

The bands obtained in continual reactions during preparation of DIG-labelled probes (section 2.5.2), were cut from the gel using a clean sterile blade under UV light. The gel fragments were placed in a Quantum prep ‘freeze n squeeze’ gel extraction spin column (Biorad) and snap frozen in liquid nitrogen. The tubes were then warmed lightly and spun at 13,000 rcf for 5 minutes. The DNA was collected at the bottom of the tube with the agarose retained in the filter. An aliquot (7μL) of eluted DNA was mixed with 3.2 pmol of one of the primers used for initial amplification in the final volume of 15 μL and submitted for sequencing to the Massey University Genome service. The sequences were compared to the sequences available in the public databases using BLAST algorithms (NCBI).

2.4.4 Hybridisation with specific probes

Hybridisation was carried out using the DIG High Prime Labelling and Detection starter Kit II (Roche Applied Science) according to manufacturer’s instructions. DIG Easy Hyb working solution was preheated and 10 ml/100 cm2 of membrane was used

Figure 1 Gel electrophoresis photo showing the incorporation of DIG-11-dUTP into PCR products. Lane L: Gene Ruler DNA ladder (Fermentas Life Sciences)Lanes 1: EHV-2 without DIG-11-dUTP (Control) Lane 2: EHV-2 with DIG-11-dUTP Lane 3: EHV-5 control Lane 4: EHV-5 with DIG-11-dUTP Lane 5: EHV-1 control Lane 6: EHV-1 with DIG-11-dUTP Lane 7: EHV-4 control Lane 8: EHV-4 with DIG-11-dUTP Lane 9: ERAV control Lane 10: ERAV with DIG-11-dUTP Lane 11: ERBV control Lane 12 ERBV with DIG-11-dUTP.

L 1 2 3 4 5 6 7 8 9 10 11 12

1031bp 800bp

for pre-hybridization of the membrane for 30minutes at the appropriate hybridisation temperature (Table 2). The appropriate DIG labelled probe was denatured by heating for 5 minutes and cooled on ice before being added to preheated DIG Easy Hyb working solution at a concentration of 2 μl/ml. The pre- hybridization solution was poured off the membrane, replaced with

probe/hybridization mixture and incubated overnight in hybridization bags in a water bath weighted down with a glass plate and gently agitated at the hybridization temperature (Table 2).

2.4.5 Optimization of hybridization conditions

The starting hybridisation temperatures were calculated based on the formula in the manufacturer’s instructions (Roche).

Tm = 49.82 + 0.41 (%G+C) – (600/L) Topt = Tm – 20-25°C

The calculated hybridization temperatures were used as a starting point for optimization. The hybridisation temperature was increased from the calculated hybridization temperature until a clear signal from the virus specific product was observed with minimal cross reaction with a similar virus (EHV-1 and EHV-4, EHV-2 and EHV-5, ERAV and ERBV) and with equine DNA. Table 2 shows the optimised temperatures used for individual viruses for detection by dot blot hybridisation.

Table 2 Hybridization temperatures for virus specific DIG Labelled DNA Probes with virus specific PCR membranes

Virus Optimised Hybridization Temperature EHV-1 57°C EHV-4 59°C EHV-2 55°C EHV-5 55°C ERAV 52°C ERBV 51°C

Table 3 A layout of a dot blot used for optimisation of the hybridisation

temperatures for EHV-2 and EHV-5 probes. Results expected from hybridisation with the EHV-5 probe are shown.

EHV-5 Probe EHV-5 primers + EHV-5 template EHV-5 primers + EHV-2 template Equine DNA EHV-2 Primers + EHV-5 template EHV-2 primers + EHV-2 template Equine DNA

* Black indicating the expected positive result

The dot blots for optimisation of the hybridization temperatures for EHV-2 and EHV- 5 probes were identical and included amplification products of PCR with either EHV- 2 or EHV-5 primers, each with either EHV-2 or EHV-5 template. In addition, 1 μl of equine DNA was also spotted on each membrane (Table 3). The layout of dot blots used for optimisation of the hybridisation temperatures for EHV-1 and EHV-4 probes is shown in Table 4.

Table 4 A layout of a dot blot used for optimisation of the hybridisation

temperatures for EHV-1 and EHV-4 probes. Results expected with the EHV-1 probe are shown. EHV-1 Probe EHV-1/4 primers + EHV-1 template EHV-1/4 primers + EHV-4 template Equine DNA EHV-1 Primers + EHV-1 template EHV-1 primers + EHV-4 template Equine DNA EHV-4 primers + EHV-1 template EHV-4 primers + EHV-4 template Equine DNA * Black indicating the expected positive result

2.4.6 Detection of DIG labelled probes

Probe/ Hybridization mixture was poured off and the membrane washed twice, in 2x SSC and 0.1% SDS at room temperature for 5 minutes. The membrane was then washed twice for 15 minutes in 0.5x SSC and 0.1% SDS at 65°C under constant agitation. The membrane was rinsed briefly in washing buffer and incubated for 30 minutes in blocking solution consisting of 10x blocking reagent (Roche) diluted 1:10

with Maleic acid buffer. Anti-Dioxigenin-AP (75m U/mL) was centrifuged for 5

minutes at 10,000 rpm and diluted 1:10,000 in blocking solution. The membrane was then incubated in 20 mL of diluted Anti-Dioxigenin-AP for 30 minutes. After washing twice for 15 minutes in 100 mL of washing buffer (0.1 M Maliec acid, 0.15 M NaCl; pH 7.5; 0.3% Tween 20)the membrane was equilibrated in 20 mL of detection buffer (0.1 M Tris-HCl, 0.1 M NaCl; pH 9.5) for 5 minutes. The membrane was then placed DNA face side up in a hybridisation bag and 1 mL of CSPD, was applied. After 5 minutes incubation in the dark, the membrane was exposed to the x-ray film for 25 minutes and developed using an automated x-ray film developer.

2.4.7 Re-probing

The membrane was rinsed thoroughly in double distilled water and washed twice for 15 minutes at 37°C in 0.2M NaOH containing 0.1% SDS to remove the DIG-labelled probe. The membrane was then rinsed thoroughly for 5 minutes in 2x SSC. The membrane was stored in 2 x SSC solutions and was not allowed to dry before re- probing. Re-probing was completed as described in section 2.5.4.