Merck Millipore Apoptag ISOL Type I/II Detection Kit

The ApopTag ISOL Fluorescence Apoptosis Detection (DNase Types I & II) technique is based upon the biochemical specificity of the enzymes T4 DNA ligase and Vaccinia Virus DNA Topoisomerase I and the unique, dual labeled, dual hairpin oligonucleotide (8,9,10). This self-annealing oligo contains two sets of complementary base sequences that spontaneously form two duplex segments resulting in a dual hairpin secondary structure (Figure 1). The oligo also contains two internal fluorescent labels which are located at opposite poles of the dual hairpin structure. At one pole is a FAM internal label and the other pole contains a CR590 label. The basis of the detection mechanism relies on the 5'-CCCTT-3' Topoisomerase I recognition site located in the middle of the dual hairpin structure. The Topoisomerase I cuts the DNA at the 3' end of the recognition site causing the dual hairpin oligo to dissociate into two separate differentially labeled hairpin oligonucleotides. The Toposiomerase I remains covalently bound to the oligo containing the recognition site and the FAM label while the other CR590 containing oligo dissociates. Hence, two differentially labeled hairpin oligo probes are created. The biochemical specificity of the provided enzymes impacts the detection aspect of the protocol, in that Vaccinia Topoisomerase I will recognize and ligate the FAM oligo to 5'-OH 3'-PO4 groups (DNase type II specific cut) whereas T4 DNA Ligase will recognize and ligate the CR590 labeled oligo to 5'-PO4 3'-OH groups (DNase type I specific cut). The ISOL Kit does not label nicks, gaps, single-stranded DNA, 3'-recessed ends or 3'-overhanging ends.