A diagram of CNVplex® technology. Two reference loci (R1/R2) and four target genomic segments (T1–T4) with various copy numbers are illustrated in this figure. They are mixed with probes, denatured, and annealed under certain conditions. The 5’ probes contain oligonucleotides specific to 5’ universal primers, and the 3’ probes include a locus discrimination linker sequence (LDLS) and oligonucleotides complementary to 3’ universal primers. Each pair of the probes contains a hybridization oligonucleotide that is specific to the human genomic DNA and can hybridize immediately adjacent to each other without a gap. The lengthening ligation system, which consists of a pair of lengthening ligation probes and a certain input template, is shown as orange lines. The probes are specific to the input template sequence and can hybridize to the template. After hybridization, probes specific to human genomic DNA and the input template are ligated at the same time using a thermally stable ligase enzyme. The PCR primers subsequently amplify the double-ligated probe, which becomes a single contiguous molecule. Amplicons with different sizes are separated by capillary electrophoresis, and the peak heights of samples are calculated and subsequently normalized to reference segments. Using LDLSs of various length and four 5’ universal primers labeled with a different fluorescent dye, CNVplex® allows 96 loci to be detected in a single PCR.