1 Introduction

Probes labeled with biotin have been used for hybridizations to Southern blots and to chromosomes in situ since the early 1980s (1). Nowadays, the labeling of double-stranded DNA probes with biotin is made easy by the availability of labeling kits that provide everything required for nick translation of DNA probes in the presence of biotinylated UTP. The method described here is based on the use of the BRL (Bethesda Research Laboratories, Gaithersburg, MD) BIONICK Labeling Kit (catalog no. 8247SA). While the labeling reaction is taking place, a fractionation column is prepared to purify the biotin-labeled DNA. After the fractions have been collected, incorporation is assessed on test strips using a BRL detection kit (catalog no. 8239SA). The whole procedure takes a whole day, with a convenient break when the test strips are incubating in the vacuum oven (see step 18 below). Several probes may be labeled in one experiment, although four probes is about the optimum number to handle by a single worker.

2 Materials

  1. 1.

    BRL BIONICK™ Labeling kit (no. 8247SA). This kit contains : 250 µL 10X dNTP mix, 250 µL DNA polymerase I, 250 µL stop buffer, 20 µL control DNA, 1 mL stop buffer.

  2. 2.

    BRL DNA Detection kit (no. 530-8239SA). This kit contains: 120 µL BRL streptavidin, 60 µL biotinylated calf intestinal alkaline phosphatase, 660 µL nitro-blue tetrazolium (NBT), 500 µL 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 25 pg/µL biotinylated DNA.

  3. 3.

    Silanized glassware and Eppendorf microcentrifuge tubes. Fill a glass beaker containing Eppendorfs with “Repelcote” (a silicon treatment water-repellent from BDH) (N.B. this material is harmful and the procedure should be carried out in a fume-hood). Swirl around, discard it, and repeat the treatment. Wash the Eppendorf tubes several times with water before autoclaving and drying in a 80°C oven. Carry out the same procedure for glassware, except after treatment, air-dry, and sterilize in an oven at 180°C.

  4. 4.

    20X SSC stock solution: 100.2 g trisodium citrate, 175.0 g sodium chloride, 1.0 L double-distilled water, autoclave.

  5. 5.

    IX SSC + 0.1% SDS: 5 mL 20X SSC, 0.1 g SDS, 95 mL double-distilled water, autoclave.

  6. 6.

    Sephadex G-50: 1 g Sephadex G-50 per 50 mL IX SSC + 0.1% sodium dodecyl sulfate (SDS).

  7. 7.

    20 mM Tris, 5 mM EDTA: 20 mL 1M Tris-HCl, pH 7.5, 10 mL 0.5M EDTA, 970 mL double-distilled water, autoclave.

  8. 8.

    Buffer 1: 6.25 mL 1M Tris-HCl, pH 8.0, 6.25 mL lM NaCl, 0.5 mL 1M MgCl2 0.125 mL Triton X-100, 237 mL sterile distilled water. Make this just before use (see Section 3., step 19).

  9. 9.

    Buffer 2: 0.75 g Bovine serum albumin (BSA) in 25 mL of buffer 1. Make this just before use (see Section 3., step 19).

  10. 10.

    Buffer 3: 20 mL 1M Tris-HCl, pH 9.5, 20 mL 1M NaCl, 10 mL 1M MgCl2 150 mL sterile distilled water. Make this just before use (see Section 3., step 19).

  11. 11.

    TE buffer (pH 8.0): 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

  12. 12.

    Deionized formamide: 200 mL formamide, 1 g Dowex 1 (chloride form) (base anion), 1 g Dowex 50W (hydrogen form). Stir for 1 h. Filter through Whatman filter paper no. 1.

  13. 13.

    NBT and BCIP are supplied in the detection kit but can also be made up as follows: NBT, 75 mg/mL in 70% dimethylformamide; BCIP, 50 mg/mL in dimethylformamide.

  14. 14.

    Bio-Rad (Richmond, CA) Bio Dot Blotting nitrocellulose membrane (or similar) material, e.g., nylon-based hybridization membranes.

  15. 15.

    Dye mix: 7.5 mL Buffer 3, 33 µL NBT, and 25 µL BCIP (NBT and BCIP are in the detection kit). Make this just before use and keep in the dark.

  16. 16.

    3M Sodium acetate: Add acetic acid to adjust pH to 6.0.

  17. 17.

    Absolute ethanol. Store at −20°C.

3 Method (see Note 1)

  1. 1.

    Keeping the contents of the BIONICK kit on ice until they are required, pipet the following into a 1.5-mL silanized Eppendorf (see Note 2): 5 µL 10X dNTP mix, 3–4 µL probe DNA (see Note 3). Make up the volume to 45 uL by adding autoclaved H2O from the kit and add 5 µL of the 10X enzyme mix provided.

  2. 2.

    Whirlimix the contents of the Eppendorf tube and centrifuge in a bench-top microfuge at 15,000g for 5 s.

  3. 3.

    Incubate the reaction mix at 16°C for 1 h in a water bath in a cold room.

  4. 4.

    While the labeling reaction is proceeding prepare a fractionation column (steps 5–8) and a test strip (step 9) for each probe being labeled.

  5. 5.

    Take a 1-mL syringe. Plug the bottom with silanized glasswool to a depth of about a 0.1 mL fraction. Do not pack the bottom too tightly.

  6. 6.

    Add Sephadex G-50 equilibrated with IX SSC + 0.1% (w/v) SDS using a Pasteur pipet. Add the first sephadex quickly, tilting the syringe, then top up carefully to avoid air bubbles, which will cause uneven flow. Continue adding small amounts until the column is equilibrated and no more drops come out from the base.

  7. 7.

    Clamp the syringe to a retort stand ready for use.

  8. 8.

    Number six siliconized Eppendorf tubes and place in a collection rack ready for use (see Note 4).

  9. 9.

    Now prepare a test strip for each probe. Using the backing paper to avoid directly handling the nitrocellulose, take a sheet of nitrocellulose. Cut a 1-cm strip and, with a sharp pencil, divide the strip into six 1-cm squares. Number each square in the top right hand corner. Label the date and probe code in the first square (note, the first sample is discarded in step 16). Place the test strip(s) on one side ready for use.

  10. 10.

    After the 1 h incubation, add 5 µL of stop buffer from the BIONICK kit to the labeling reaction and mix briefly. Place the Eppendorf tube on ice.

  11. 11.

    Add 15 µL Dextran blue to each Eppendorf tube. Mix well and then pipet all the mixture to the top of the fractionation column.

  12. 12.

    Quickly add 100 µL IX SSC + 0.1% SDS to the top of the column, washing out the Eppendorf tube first and then adding the remainder to the column. Allow the drops coming from the column to collect into a beaker and discard them.

  13. 13.

    When the drops stop, place the rack of six Eppendorf tubes below the column so that the first one is directly beneath the syringe nozzle.

  14. 14.

    Add 100 µL of IX SSC + 0.1% SDS to the top of the column and let the drops collect into the first Eppendorf tube. There should be about five to six drops.

  15. 15.

    Move the rack until the second Eppendorf tube is underneath the syringe and add another 100 µL of IX SSC + 0.1% SDS to the top of the column.

  16. 16.

    Continue until all the blue band has moved off the column and all the Eppendorf tubes have been used (see Note 5). Discard the first tube.

  17. 17.

    Lay out the test strip on the bench. Using a clean pipet tip each time, remove a 1-µL aliquot from each collected fraction and spot it into the center of the correspondingly numbered square on the test strip.

  18. 18.

    Bake the filters at 80°C for 1 h in a vacuum oven.

  19. 19.

    Prepare the buffers 1 to 3 for the detection of biotin.

  20. 20.

    After baking the test strip, rehydrate for 1 min in buffer 1 at room temperature.

  21. 21.

    Incubate the strip for 20 min in buffer 2 at 42°C (see Note 6).

  22. 22.

    Blot the strip dry between two sheets of filter paper and dry it in the vacuum oven at 80°C for 10–20 min.

  23. 23.

    Rehydrate the strip in buffer 2 for 10 min.

  24. 24.

    Incubate the strip in 2 mL of buffer 1 + 4 µL streptavidin (from the kit) for 10 min with gentle agitation.

  25. 25.

    Wash the strip in 40 mL of buffer 1 for 3 min. Repeat this step twice.

  26. 26.

    Incubate the strip for 10 min with 2 mL buffer 1 + 2 µL kit poly alkaline phosphatase.

  27. 27.

    Wash the strip with 40 mL of buffer 1. Repeat this step.

  28. 28.

    Wash with 40 mL of buffer 3 for 3 min. Repeat this step.

  29. 29.

    Prepare a silver foil-covered small tube.

  30. 30.

    Add the dye mix.

  31. 31.

    Incubate the strip in the dye solution in the foil-wrapped tube for 30 min or until test strip is fully developed (see Note 7).

  32. 32.

    Wash the strip with 20 mM Tris, 5 mM EDTA to terminate the reaction.

  33. 33.

    Dry the test strip in the vacuum oven at 80°C for 1–2 min.

  34. 34.

    Based on the results of the test strip (see Notes 7 and 8) combine all the labeled fractions you wish to keep into one tube. Usually about four to five fractions are kept (which equals 400–500 µL in total).

  35. 35.

    Add 1/10 vol of 3M sodium acetate (40–50 µL).

  36. 36.

    Add 2 vol of cold absolute alcohol (about 1 mL) and mix by inverting the Eppendorf tube.

  37. 37.

    Freeze the mixture at −70°C for 15 min or at −20°C for 2 h.

  38. 38.

    Centrifuge the sample at 15,000g for 10 min and carefully remove the supernatant.

  39. 39.

    Dry the pellet by placing the tube in a desiccator connected to a vacuum pump for 5 min.

  40. 40.

    Resuspend the pellet in 20 µL of deionized formamide and keep it in the refrigerator at 4°C if it is to be used the next day, or resuspend it in 20 µL of TE buffer and store at −20°C.

4 Notes

  1. 1.

    For safety reasons, wear gloves throughout the whole labeling procedure.

  2. 2.

    It is essential when handling biotin-labeled probes that all glassware and Eppendorf microtubes are silanized.

  3. 3.

    The probe DNA can be in a plasmid vector or used as the excised insert. The concentration should be 1 µg/µL.

  4. 4.

    Standard Eppendorf racks can be used.

  5. 5.

    Usually the bulk of the fraction (visible as a blue band) passes through the column in the third to the fifth Eppendorf tube. If you have packed the silanized wool too tightly, it may take longer to pass through and additional Eppendorf tubes may be required.

  6. 6.

    If you are using nylon membranes and not nitrocellulose, incubate at 65°C.

  7. 7.

    The intensity of color in the spot on the test strip is a good indication of labeling incorporation. Keep only those fractions which gave strong color spots on the strip (usually three to four fractions). Estimations of the levels of incorporation can be achieved by co-incubating a strip with serial dilutions of control biotinylated DNA provided in the kit (see kit instructions). However, we have found that provided the color is strong after 30 min to 1 h, there is sufficient incorporation for successful in situ hybridization and we do not routinely estimate incorporation levels.

  8. 8.

    If the spots fail to develop any color, check the concentration of your probe DNA and that the NBT and BCIP have not crystallized in the stock bottles (if so, replace with fresh samples).