DIAPOPS Procedure

  1. Covalent Binding of Solid Phase Primer
  2. Amplification
  3. Detection
  4. Wash after detection to prepare for rehybridization or storage
  5. Rehybridization

1. Covalent Binding of Solid Phase Primer

  1. Make sure that your solid phase primer is phosphorylated at the 5'-end ( Phosphorylation of the solid phase primer) and that a linker of at least 10 T's** (Thymidine's) is added to the 5'-end of the primer ( DIAPOPS results as a function of a linker in the 5'-end of the solid phase primer).
  2. Prepare a coating mix consisting of ( Buffers for covalent binding of solid phase primer) : One ng/µl solid phase primer and 10 mM ( Figure 4: Results from optimization of the procedure for covalent binding of the solid phase primer to the surface of NucleoLink) EDC ( EDC) (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) in 10 mM 1-methylimidazole (1-MeIm) ( Reaction of the carbodiimide activated DNA with 1-methylimidazole) (pH 7.0).
  3. Add 100 µl of this freshly prepared coating mix to each well ( Results from stability studies on the mix of primers and EDC before addition to the NucleoLink Strips). This gives a total of 100 ng 5’-phosphorylated solid phase primer added to each well ( Concentration of the phosphorylated oligonucleotide).
  4. Seal the NucleoLink Strips (e.g. with Nunc Sealing Tape, Cat. No. 236366) ( Evaporation during incubation).
  5. Incubate the NucleoLink Strips at 50ºC for 4-24 hours ( Time of incubation during the covalent binding).
  6. Wash the empty NucleoLink wells three times ( Wash after binding of the solid phase primer to the surface of the NucleoLink Strips) with freshly prepared 0.4 M NaOH and 0.25% Tween 20 ( 0.4 M NaOH and 0.25% Tween 20 (1000 ml)), pre-warmed to 50ºC (it is possible to prepare the NaOH in advance and add the Tween 20 just before use).
  7. Soak for 15 minutes at 50ºC with freshly prepared 0.4 M NaOH and 0.25% Tween 20, pre-warmed to 50ºC.
  8. Wash three times with freshly prepared 0.4 M NaOH and 0.25% Tween 20, pre-warmed to 50ºC. Empty the strips thoroughly ( Emptying the NucleoLink Strips).
  9. To remove NaOH residues, wash the empty NucleoLink wells three times, soak for 5 minutes and wash three times, all with distilled water at Room Temperature (RT) ( Wash after binding of the solid phase primer to the surface of the NucleoLink Strips). Empty the Strips.
  10. The coated and washed, empty, NucleoLink Strips can be stored at 4ºC or below in an polythene bag ( Storage of NucleoLink Strips). The Strips should not be sealed ( Sealing during storage).

2. Amplification

  1. To block the wells before amplification ( Pre-PCR blocking), add to each well 200 µl of DIAPOPS buffer with 10 mg/ml BSA ( DIAPOPS buffer with 10 mg/ml BSA (100 ml)).
  2. Shake at RT for 1 hour ( Difference in BSA blocking conditions).
  3. Empty the strips. No further washing is necessary, but it is important to completely empty the wells ( Milky white PCR liquid in DIAPOPS after thermal cycling). The strips cannot be stored after this blocking step ( Accelerated storage experiment with BSA blocking before storage).
  4. Add PCR* mix ( PCR mix) to the wells (normally 20 µl or 45 µl). The concentration of the two primers in the liquid phase should be in a ratio of 1:8 ( DIAPOPS signal as a function of primer ratio) with the primer used as the solid phase primer in the lowest concentration. At the Nunc A/S Research Laboratory the concentration used is 25 pmol/reaction of the primer not used as the solid phase primer, and 25/8 pmol/reaction of the primer used as the solid phase primer. A concentration of 0.1%-0.25% Tween 20 is recommended.
  5. Add DNA template ( DNA template) to each well (the total reaction volume has been tested with both 25 µl and 50 µl) ( Shape and volume of NucleoLink Strips).
  6. Seal the NucleoLink Strips with Tape 8 ( Sealing of NucleoLink Strips during DIAPOPS and storage).
  7. Place the Strips in a thermal cycler block ( Inserting and removing NucleoLink Strips from thermal cyclers).
  8. Place the silicone spacer plate on the tape-sealed NucleoLink Strips ( Spacer Plate) and tighten the heated lid firmly ( Inserting and removing NucleoLink Strips from thermal cyclers).
  9. Program the thermal cycler with temperatures and cycling parameters specific for your system ( Factors to optimize), and start the program.
  10. Remove the NucleoLink Strips from the thermal cycler after thermal cycling ( Inserting and removing NucleoLink Strips from thermal cyclers) and empty the Strips. The liquid phase can be stored in GeNuncTM wells (GeNuncTM 120, Cat. No. 232549), sealed with tape (Nunc Sealing Tape, Cat. No.: 236366) at 4ºC in a sealed polythene bag.
  11. Wash ( Wash after PCR) the empty NucleoLink Strips three times ( The term: Wash three times ), soak for 5 minutes, and wash three times to denature the solid phase product ( Denaturation of solid phase product), all with freshly made 0.2 M NaOH and 0.1% Tween 20 ( 0.2 M NaOH, 0.1% Tween 20 (1000 ml)) at RT.
  12. Wash ( Wash after PCR) the empty NucleoLink wells three times, soak for 5 minutes, and wash three times, all with DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) at RT.

3. Detection

  1. Add 100 µl of 50-100 nM ( DIAPOPS signal as a function of probe concentration) biotinylated hybridization probe ( Hybridization detection of solid phase product) diluted in 5 x SSC, 0.1% Tween 20 and 0.5% Blocking reagent (BR) (Cat. No.: 1096176. Boehringer Mannheim, Mannheim, Germany) ( 50 nM biotinylated hybridization probe diluted in 5 x SSC, 0.1% Tween 20 and 0.5% blocking reagent (BR) (10 ml)) to each well.
  2. Seal the NucleoLink Strips with Tape (Nunc Sealing Tape, Cat. No. 236366) ( Evaporation during incubation) and incubate at 45-50ºC for one to 20 hours (probe dependent) ( Hybridization detection of the solid phase PCR product).
  3. Wash ( Wash after hybridization) the empty NucleoLink wells three times at RT with 0.5 x SSC and 0.1% Tween 20 ( 0.5 x SSC, 0.1% Tween 20 (1000 ml)).
  4. Soak for 15 minutes at 50ºC ( Hybridization detection of the solid phase PCR product) with 0.5 x SSC and 0.1% Tween 20.
  5. Wash ( Wash after hybridization) three times at RT with 0.5 x SSC and 0.1% Tween 20.
  6. Detection of biotin-label on the hybridized probe ( Hybridization detection of solid phase product) :
    1. When using Alkaline phosphatase (AP) ( Alkaline Phosphatase (AP)) : Add to each well 100 µl AP conjugated streptavidin (Code no. D0396 - DAKO, Glostrup, Denmark) diluted 1:3000 (or as the producer suggests) in DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20).
    2. When using horse radish peroxidase (HRP) ( Horse-Radish Peroxidase (HRP)) : Add to each well 100 µl HRP conjugated streptavidin (Code no. P-0397. DAKO, Glostrup, Denmark) diluted 1:5000 (or as the producer suggests) in DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20).
  7. Incubate for one hour at 37ºC - 50ºC ( Incubation with streptavidin-alkaline phosphatase conjugate) sealed with Nunc Sealing Tape ( Evaporation during incubation).
  8. Wash ( Wash after addition of enzyme conjugate) the empty NucleoLink Strips three times, soak for 5 minutes and wash three times, all with DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) at RT.
  9. Two substrates have been tested with AP ( Alkaline Phosphatase (AP)) ; 4-MUP (4 methylumbelliferyl phosphate) ( 4-Methylumbelliferyl Phosphate (4-MUP)) and pNPP (para nitrophenylene phosphate) ( para-Nitro Phenyl Phosphate (pNPP)). One color-forming reagent has been tested with HRP ( Horse-Radish Peroxidase (HRP)) ; TMB (3,3',5,5'-tetramethylbenzidine) ( 3,3',5,5'-tetramethylbenzidine (TMB)) in a ready-to-use solution (Cat. No.: 4380H. Kem-En-Tec, Copenhagen, Denmark).
    1. When using 4-MUP: Add 100 µl of 1 mM 4-MUP dissolved in 1 M diethanolamine (pH 9.8) and 1 mM MgCl2 ( 1 mM 4-MUP dissolved in 1 M diethanolamine (pH 9.8), 1 mM MgCl<font size=1>2</font> (1000 ml)) to each well.
    2. When using pNPP: Add 100 µl of 1 or 10 mg/ml pNPP in 1 M diethanolamine (pH 9.8) and 1 mM MgCl2 ( PNPP in 1 M diethanolamine (pH 9.8) and 1 mM MgCl2) to each well.
      1. When using TMB: Add 100 µl of the ready-to-use solution to each well.
  10. Substrate incubation ( Summary).
    The NucleoLink Strips should be sealed with Nunc Sealing Tape when incubating for longer than 30 minutes ( Evaporation during incubation).
    1. When using 4-MUP, incubate at 37-50ºC in the dark for 30-60 minutes. Add 50 µl of 3 M K2HPO4 ( 3 M K2HPO4 (500 ml)) to stop the hydrolyzation of 4-MUP.
    2. When using pNPP, incubate at RT for 30 minutes (10 mg/ml) to 24 hours (1 mg/ml) ( para-Nitro Phenyl Phosphate (pNPP)). Add 100 µl of 1 M NaOH ( 1 M NaOH (1000 ml)) to stop hydrolyzation of pNPP.
    3. When using TMB, incubate for 30 minutes at RT. Add 100 µl 0.1 M H2SO4 ( 0.1 M H2SO4 (1000 ml)) to stop the reaction. Observe: Rehybridization ( Rehybridization to the solid phase PCR product) is not possible after addition of acid.
  11. To measure the signal ( Summary of enzymes and substrates tested in DIAPOPS).
    1. For detection of hydrolyzed 4-MUP ( 4-Methylumbelliferyl Phosphate (4-MUP)), determine the signal in fluorescence plate reader: Excitation wavelength 360 nm, emission wavelength 450 nm (also if the reaction has been stopped with K2HPO4).
    2. For detection of hydrolyzed pNPP ( para-Nitro Phenyl Phosphate (pNPP)), measure OD in a normal ELISA plate reader at 405 nm (also if the reaction has been stopped with NaOH).
    3. For detection of TMB ( 3,3',5,5'-tetramethylbenzidine (TMB)), measure OD in a normal ELISA plate reader at 450 nm after the reaction has been stopped with acid (if the reaction is not stopped with acid, the color is blue and can be measured at 655 nm).

4.Wash after detection to prepare for rehybridization ( Rehybridization to the solid phase PCR product) or storage ( Storage of NucleoLink Strips after PCR with immobilized amplicons)

  1. Remove substrate from wells after measuring the catalyzed substrate.
  2. Wash the empty NucleoLink wells three times, soak for 5 minutes and wash three times with freshly made 0.2 M NaOH, and 0.1% Tween 20 ( M NaOH, 0.1% Tween 20 (1000 ml)) at RT.
  3. Wash the empty NucleoLink Strips three times, soak for 5 minutes and wash three times with distilled water or DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) at RT. Empty the strips thoroughly ( Emptying the NucleoLink Strips).
  4. The washed NucleoLink Strips can be stored empty at 4ºC or below in an polythene bag ( Storage of NucleoLink Strips after PCR with immobilized amplicons). The Strips should not be sealed ( Sealing during storage).

5. Rehybridization ( Rehybridization to the solid phase PCR product)

  1. No additional treatment of the NucleoLink Strips is necessary after storage. Commence with step 1 in the detection section and add hybridization solution directly to the empty, dry, NucleoLink Strips.

*The Polymerase Chain Reaction (PCR) process is covered by US patents owned by Hoffmann-La Roche, Inc and F. Hoffmann-La Roche Ltd.
**Use of a polythymidine linker in solid phase amplification is covered by patents owned by GenSet.