1 Introduction

Congenital stationary night blindness (CSNB) is a rare heterogeneous retinal disease that is present at birth. Clinically the disease is characterized by vision impairment under dim light conditions and can be associated with other ocular defects as myopia, nystagmus, strabismus, and reduced visual acuity (1). Different forms of CSNB are classified according to their mode of inheritance, phenotype, and mutated genes (2). CSNB is caused by mutations in genes encoding different components of the phototransduction cascade or proteins involved in signaling from photoreceptors to adjacent neurons (1).

To date, more than 10 different genes have been associated with CSNB (3, 4). Most of the patients with mutations in these genes show a typical electrophysiological phenotype characterized by an electronegative waveform of the dark-adapted bright flash electroretinogram (ERG), in which the amplitude of the b-wave is smaller than that of the a-wave (5). According to this so-called Schubert-Bornschein type of ERG response CSNB can be divided into two subtypes—incomplete (ic) and complete (c) CSNB (2). icCSNB has been characterized by both a reduced rod b-wave and substantially reduced cone responses due to both ON- and OFF-bipolar cell dysfunction, while the complete type is associated with a drastically reduced rod b-wave response due to ON-bipolar cell dysfunction, but largely normal cone b-wave amplitudes (6). icCSNB has been associated with mutations in CACNA1F (7, 8), CABP4 (9), and CACNA2D4 (10), which encode for proteins forming the alpha subunit of a calcium channel, for a calcium-binding protein which interacts with this subunit, and for an auxiliary subunit of this channel, respectively. The latter one is most likely important for the correct localization of the alpha subunit. These proteins are located at the synapses of photoreceptor cells. cCSNB has been associated with mutations in NYX (11, 12), GRM6 (13, 14), and TRPM1 (3, 15, 16)—proteins expressed in bipolar cells and which are important for the further signaling in the retina (1, 3).

The majority of mutations associated with CSNB have been identified in CACNA1F and NYX genes (1). Still, a number of patients lack mutations in these genes indicating that new gene defects need to be discovered.

Because of the heterogeneity of CSNB, the identification of the underlying cause of the disease is complicated (17). Clinical data does not always direct to the disease-causing mutated gene. Thus, in some cases, direct sequencing of all known genes associated with CSNB might be requested. However, this is time-consuming and cost-ineffective. In order to overcome the challenges to confirm the diagnosis of CSNB and to determine the genetic defect an arrayed primer extension (APEX)-based microarray for the analysis of genes associated with CSNB has been developed (17).

APEX is a genotyping method that allows detection of hundreds of known variations in the genome in a single multiplexed reaction (18, 19). The APEX reaction involves the target DNA hybridization to the sequence-specific oligonucleotides immobilized on a glass slide followed by an enzymatic single base extension reaction in which DNA polymerase incorporates a dye-labelled terminator nucleotide to the hybridized oligonucleotide.

For the mutation detection by APEX method the DNA regions of interest are first amplified by polymerase chain reaction (PCR). The amplified products are concentrated and purified with PCR purification columns. The fragmentation of purified products and functional inactivation of the unincorporated residual dNTPs are achieved by shrimp alkaline phosphatase (sAP) and Uracil DNA-Glycosylase (UNG) treatment. Fragmented and denatured PCR products are used for the primer extension reaction on the microarray. A schematic overview of the APEX-based mutation detection on a microarray is presented in Fig. 1.

Fig. 1.
figure 00191

Schematic overview of the APEX method. The DNA regions of interest are first amplified by PCR. In order to achieve the fragmentation of the PCR products with uracile-DNA glycosylase a fraction of dTTPs are substituted with dUTPs in the reaction mixture. The fragmented DNA is mixed with a thermostable DNA polymerase and fluorescently labelled ddNTPs for APEX reaction. After the single base extension reaction and washing steps, the microarray signals will be detected and analyzed. WT wild type, MUT mutation.

Full size image

APEX array for the analysis of CSNB-associated genes is a robust, cost-effective, and easily modifiable tool for a first-pass genetic testing (17). Current Asper Biotech CSNB microarray covers 159 mutations from 11 genes—RHO (MIM 180380), PDE6B (MIM 180072), GNAT1 (MIM 139330), CABP4 (MIM 608965), GRM6 (MIM 604096), SAG (MIM 181031), NYX (MIM 300278), CACNA1F (MIM 300110), CACNA2D4 (MIM 608171), GRK1 (MIM 180381), and TRPM1 (MIM 603576). This microarray is regularly updated to cover as many known mutations associated with CSNB as possible.

2 Materials

2.1 Polymerase Chain Reaction

  1. 1.

    10× PCR buffer: 750 mM Tris–HCl (pH 8.8 at 25°C), 200 mM (NH4)2SO4, 0.1% Tween 20.

  2. 2.

    25 mM MgCl2.

  3. 3.

    dNTP mix: 2.5 mM of dATP, dCTP, dGTP, 2 mM of dTTP, and 0.5 mM of dUTP.

  4. 4.

    PCR primers (Metabion GmbH, Germany).

  5. 5.

    Smart-Taq Hot DNA Polymerase (10 U/μl) (Naxo, Estonia).

  6. 6.

    1× Tris–borate–EDTA (TBE) Buffer: 89 mM Tris–Borate, 10 mM EDTA (pH 8.3).

  7. 7.

    1.5% TBE agarose gel with ethidium bromide: For a 1.5% agarose gel, add 1 gram of agarose to 100 ml of 1× TBE buffer. Place the gel solution into the microwave oven, boil, and swirl the solution until agarose particles are dissolved. Cool the gel to 60°C, add ethidium bromide (final concentration 0.5 μg/ml). Pour the gel into the gel casting tray, insert the comb into the gel, and let the gel set about 30 min.

  8. 8.

    DNA loading dye.

  9. 9.

    Thermal cycler.

  10. 10.

    ddH2O.

2.2 Column Purification

  1. 1.

    GenJet™ PCR Purification Kit (Fermentas, Lithuania).

2.3 Fragmentation

  1. 1.

    UNG 1 U/μl.

  2. 2.

    sAP 1 U/μl.

  3. 3.

    10× UNG buffer: 500 mM Tris–HCl (pH 9.0), 200 mM (NH4)2SO4.

  4. 4.

    1.5% TBE agarose gel with ethidium bromide and 1× TBE Buffer.

2.4 APEX

  1. 1.

    CSNB microarray slides (Asper Biotech Ltd., Estonia).

  2. 2.

    Thermo Sequenase™ DNA Polymerase 32 U/μl (GE Healthcare, UK).

  3. 3.

    Thermo Sequenase™ Reaction Buffer: 260 mM Tris–HCl (pH 9.5) and 65 mM MgCl2.

  4. 4.

    Thermo Sequenase™ Dilution Buffer: 10 mM Tris–HCl (pH 8.0), 1 mM 2-mercaptoethanol, 0.5% Tween 20 (v/v), 0.5% Nonidet P-40 (v/v).

  5. 5.

    Fluorescently labelled ddNTPs: 50 μM Cy3-ddCTP, 50 μM Texas Red-5-ddATP, 50 μM Fluorescein-12-ddGTP, 50 μM Cy5-ddUTP.

  6. 6.

    LifterSlip™ cover slides 22  ×  32 mm2 (Erie Scientific Company, NH, USA).

  7. 7.

    0.3% Alcanox detergent solution (Alcanox Inc., NY, USA).

  8. 8.

    Atlas Antifade (BioAtlas, Estonia).

  9. 9.

    Genorama QuattroImager™ (Genorama Ltd., Estonia).

  10. 10.

    Thermal plate with humidified chamber.

  11. 11.

    dH2O.

3 Methods

3.1 Polymerase Chain Reaction

  1. 1.

    Amplify the DNA regions flanking CSNB mutations by PCR in singleplex reactions. Prepare the PCR reaction mix for each PCR primer pair in a total volume of 25 μl containing 30 ng of genomic DNA, 1X PCR buffer, 2.5 mM MgCl2, 0.25 mM dNTP mix with dUTP (see Note 1), 5 pmol of forward and reverse primer, 1 U Smart Taq Hot DNA Polymerase, and ddH2O.

  2. 2.

    Conduct the PCR amplification in a thermal cycler under the following conditions: 95°C for 15 min; 9 cycles at 95°C for 20 s, 68°C for 20 s (−1°C per cycle), and 72°C for 40 s; 18 cycles at 95°C for 20 s, 58°C for 20 s, and 72°C for 40 s; 9 cycles at 95°C for 20 s, 56°C for 20 s, and 72°C for 40 s; and final extension at 72°C for 7 min.

  3. 3.

    Control the amplification efficiency by loading the samples on a 1.5% agarose gel in 1× TBE Buffer.

3.2 Concentration and Purification of the Amplified Products

  1. 1.

    Pool the PCR amplification products into 1.5 ml microcentrifuge tubes.

    For 160 μl of pooled amplification product add 160 μl of Binding Buffer mix briefly by pipetting.

  2. 2.

    Transfer the solution from step 2 into the GeneJET™ purification column. Centrifuge the column at 12,000  ×  g (10,000–14,000 rpm, depending on the rotor type) for 1 min, and discard the flow-through.

  3. 3.

    Add 700 μl of Wash Buffer to the column. Centrifuge the column at 12,000  ×  g for 1 min. Discard the flow-through and place the purification column back into the collection tube.

  4. 4.

    Centrifuge the empty column for 2 min at 12,000  ×  g to remove any residual wash buffer (see Note 2).

  5. 5.

    Insert the column into a new 1.5 ml microcentrifuge tube. Add 30 μl of Elution Buffer in the center of the column and incubate at room temperature for 1 min.

  6. 6.

    Centrifuge the column at 12,000  ×  g for 2 min. Discard the purification column.

  7. 7.

    Store the purified PCR products at −20°C.

3.3 Fragmentation and Inactivation of dNTPs

  1. 1.

    Prepare the UNG–sAP reaction mix containing 4 μl of 10× UNG Buffer, 1.2 U of UNG, and 1 U of sAP and add the mixture to 30 μl of purified PCR products.

  2. 2.

    Incubate the sample at 37°C for 1 h. For product fragmentation denature the sample for 10 min at 95°C.

  3. 3.

    Control the fragmentation efficiency by loading the samples on a 1.5% agarose gel in 1× TBE Buffer. Use non-denatured samples as controls. Fragmentation is successful if no intact denatured PCR product is visible in the gel.

3.4 APEX Reaction and Slide Imaging

  1. 1.

    Wash the CSNB APEX slides 1× in 95°C dH2O. Lift the slides slowly out of the water in order not to leave any droplets of water onto the slides (see Note 3).

  2. 2.

    Place the slides on a prewarmed thermoplate.

  3. 3.

    Prepare the APEX reaction mixture as follows:

    Tube A: 35 μl of UNG–sAP-treated and fragmented amplification products.

    Tube B: 5 μl of Reaction Buffer and 1.2 μl of each fluorescently labelled 50 μM ddNTP.

    Tube C: Dilute 3.2 U of Thermo Sequenase™ DNA Polymerase with 0.9 μl of dilution buffer.

  4. 4.

    Denature the PCR products in the tube A for 10 min at 95°C.

  5. 5.

    Meanwhile mix the content of tube C with tube B.

  6. 6.

    Centrifuge the tube A briefly and add the prepared reaction mixture (tube B  +  C) to the sample. Vortex and centrifuge briefly and apply the mixture immediately to the pre-warmed slides on a heated plate (see Note 4).

  7. 7.

    Cover the slide quickly with LifterSlip™ (see Notes 5 and 6) and incubate for 20 min at 58°C in the dark and humid chamber.

  8. 8.

    To terminate the reaction wash slides after incubation with 95°C dH2O, then in 0.3% warm Alcanox solution for 3 min, and twice with 95°C dH2O.

  9. 9.

    Remove the slides from water; apply a droplet of Atlas Antifade Reagent to minimize bleaching, and cover the slide with a coverslip.

  10. 10.

    Image the slides with Genorama QuattroImager™. Mutations are determined by using Genorama Genotyping Software™ (Genorama Ltd., Estonia).

4 Notes

  1. 1.

    20% of dTTPs are substituted with dUTPs in order to achieve the fragmentation of the PCR products during UNG–sAP treatment. Fragmentation is necessary for the APEX reaction efficiency.

  2. 2.

    This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.

  3. 3.

    The surface of silanized slides is hydrophobic and no droplets of water should stay on the glass surface if the slides are slowly lifted out of the water.

  4. 4.

    The reaction mixture should be immediately applied onto the microarray slide to assure that the DNA is denatured.

  5. 5.

    The reaction mixture applied onto microarray slide should be covered with LifterSlip™ as fast as possible to avoid evaporation.

  6. 6.

    In order to remove the bubbles that may have formed, gently bend the slides.