Human identification is commonly based on the analysis of short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), or deletion insertion polymorphisms (DIPs), depending on the demands of an examination or on the sample quality. These multiplex assays are complex systems that require a defined range of template input.
The Investigator Quantiplex HYres Kit contains reagents and a DNA polymerase for the specific amplification of two target regions and the direct detection of the specific PCR products on the Rotor-Gene Q real-time PCR cycler or Applied Biosystems 7500 Real-Time PCR Systems. The target for total human DNA is a 146 bp autosomal multi-copy region of the human genome. This quantification target was selected in order to give high sensitivity with high reliability within different individuals and populations. It is detected using the green channel on the Rotor-Gene Q or the FAM dye channel on Applied Biosystems instruments.
The target region for male DNA quantification was selected in order to reliably give the same high sensitivity within different individuals and populations and in the presence of mixed DNA samples. This target region is detected as a 129 bp fragment using the red channel on the Rotor-Gene Q or the Cy5 dye channel on Applied Biosystems instruments.
In addition, the Investigator Quantiplex HYres Kit contains a balanced internal amplification control that is used to test successful amplification and identify the presence of PCR inhibitors. This heterologous amplification system is detected as a 200 bp internal control (IC) in the yellow channel on the Rotor-Gene Q or in the VIC dye channel on Applied Biosystems instruments.
Detection of amplification is performed using Scorpions primers and a novel, fast PCR chemistry. Scorpions primers are bifunctional molecules containing a PCR primer covalently linked to a probe (see the figure "Scorpions primers and their function"). The fluorophore in this probe interacts with a quencher, also incorporated into the probe, which reduces fluorescence. During PCR, when the probe binds to the amplicon, the fluorophore and quencher become separated. This leads to an increase in fluorescence in the reaction tube.
The reaction chemistry was carefully optimized to further support the rapid mechanism. The novel PCR chemistry and enzyme, which were specifically developed for human and male DNA identification, enable high sensitivity and speed. Furthermore, the specially developed PCR chemistry contains the patent-pending additive Q-Bond, which significantly reduces denaturation, annealing, and extension times.