The DNA-label serves as template for the PCR and should be carefully designed. Most important is that the DNA-label and the primer pair used produce negligible amounts of non-specific primer-dimer products. Its length does not seem to be crucial. We have tested lengths between 66-1098 bp and found no important dependence. However, we have not performed any systematic study. Usually we design the PCR system to give short amplicons (60-150 basepairs), to have high PCR efficiency and to keep cost down. If streptavidin-biotin linkage is going to be used to connect the DNA to the detection antibody, biotinylated primers can be used to produce the DNA-label. For chemical conjugation suitably modified polynucleotides are available from most oligohouses, and the shorter they are, the less expensive. The modification can be either an amino- or a thiol-group depending on the chemistry that will be used. If succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) is used, the polynucleotide should be amino modified. We always modify the 5' end of the polynucleotide because it is cheaper, but 3' end modifications should also be fine. To minimize risk of contamination, we recommend using a different DNA sequence for real-time immuno-PCR from those used by others in the lab. Ideally a non-natural DNA sequence can be designed. Such DNA sequences including primers and protocol are available from our center (www.tataa.com).

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