Summary of enzymes and substrates tested in DIAPOPS

Introduction
Summary
Conclusion

1. Introduction
In the DIAPOPS procedure ( ) solid phase amplified DNA is detected by hybridization. The hybridization signal is enhanced using an enzymatic reaction. Two different enzymes have been tested in DIAPOPS: Alkaline phosphatase (AP) ( ), and horse radish peroxidase (HRP) ( ).

2. Summary
The substrates tested for the enzymes and the reaction conditions are listed in Table 1.

Enzyme	Substrate	Concentration	Incubation time	Incubation temperature	Wavelength	Remarks
AP	pNPP	1 mg/ml	12-24 hours	RT	405 nm	Norm. ELISA concentration
AP	pNPP	10 mg/ml	30 minutes	RT	405 nm	Improved DIAPOPS concentration
AP	4-MUP	1 mM	30 minutes	37º-50ºC	ex. 360 nm imm. 450 nm	Fluorescence
HRP	TMB	Unknown (ready-to-use solution)	30 minutes	RT	450 nm with acid 655 nm without acid	Rehybridization not possible after addition of acid

Table 1: A list of enzymes and substrates tested in DIAPOPS with reaction conditions.

3. Conclusion
Both AP ( ) and HRP ( ) will perform satisfactorily with the listed substrates when used in DIAPOPS ( ).

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