The washing steps in the DIAPOPS procedure

  1. Introduction
  2. Wash after binding of the solid phase primer to the surface of the NucleoLink Strips
  3. Wash after PCR
    1. Flushing of the washing equipment when changing washing buffers
  4. Wash after hybridization
  5. Wash after addition of enzyme conjugate
  6. Conclusion

1. Introduction
In the DIAPOPS procedure, several washing step are carried out. The importance of conducting these washes adequately ( The term: Wash three times ) is described in the following sections.

2. Wash after binding of the solid phase primer to the surface of the NucleoLink Strips
After the covalent binding reaction of the solid phase primer to the surface of the NucleoLink Strips, there will be unbound primers as well as an amount of the EDC urea derivative originating from the activation process of the phosphate group on the solid phase primer ( Figure 2). This urea derivative must be washed off, as its presence would inhibit the subsequent PCR reaction. This wash is carried out with NaOH ( DIAPOPS Procedure). After the NaOH wash, it is important also to remove residues of NaOH. Remaining NaOH will also inhibit the PCR. This wash is performed with the DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20).

3. Wash after PCR
After the PCR, the solid phase products are double-stranded. In order to be detected using hybridization, they must be rendered single-stranded ( Denaturation of solid phase product). This is carried out using NaOH ( DIAPOPS Procedure). The denaturing treatment must be carefully conducted, in order to denature all the solid phase products ( Analysis of the method for denaturation of the solid phase amplicons after PCR). If this denaturation is not carried out, detection of the solid phase products can not take place, and no DIAPOPS signals will be observed. Furthermore, it is important to wash of the NaOH used in the denaturation process. NaOH remaining in the NucleoLink Strips may decrease the efficiency of the hybridization step, and either totally destroy the DIAPOPS analysis or only diminish the DIAPOPS signals. This wash is performed with the DIAPOPS buffer ( DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20).

3 a) Flushing of the washing equipment when changing washing buffers
If a Nunc-Immuno Wash is used ( Tools for washing) it is very important to flush the washing equipment with the DIAPOPS buffer before a new wash. The wash immediately prior to the washing with the Tris-buffer has been with NaOH. If the Nunc-Immuno Wash is not flushed, the first Strips will be only washed with NaOH, which is in the washing equipment, and this will prevent an efficient hybridization.

4. Wash after hybridization
This wash is performed to remove both non-bound probes and non-specifically hybridized probes. If this wash is performed suboptimally, false positive signals may arise from non-specifically hybridized probes. The wash may be optimized for each single probe sequence ( Hybridization detection of the solid phase PCR product). Remaining non-hybridized probes are likely to be washed away during the subsequent wash after addition of the enzyme conjugate. However, if a large excess of non-hybridized probes remain they may saturate the enzyme conjugate. Consequently, the hybridized probes on the surface may not all be labeled with the enzyme.

5. Wash after addition of enzyme conjugate
The last wash removes non-specifically bound enzyme conjugate ( DIAPOPS Procedure). A failure to perform this wash optimally will yield a large background signal ( Background in DIAPOPS) in the subsequent enzyme substrate reaction. The consequence of this may be that all the true signals are masked by the background signal.

6. Conclusion
All washes in the DIAPOPS procedure should be carefully performed. Several problems may occur if any one of the washes are neglected or are conducted inadequately.