6 Indel genotyping1.6.1 Automated DNA Extraction using EZ1 Instrument and EZ1 DNA Investigator Kit OverviewEZ1 instruments and the EZ1 DNA Investigator Kit automatically purifies genomic DNA from 1″6 samples (EZ1 Advanced) or 1″14 samples (EZ1 Advanced XL). Sample input volumes of 200 јl or 500 јl can be selected while using EZ1 instruments allowing purification from different amounts of starting material. Up to 6 samples (EZ1 Advanced) or up to 14 samples (EZ1 Advanced XL) can be processed in a single run. The worktable of EZ1 instruments is where the samples and contents of the EZ1 DNA Investigator Kit are loaded.
EZ1 DNA Investigator Kit contains reagent cartridges including guanidine salt, disposable tip holders, disposable filter-tips, sample tubes (2 ml), elution tubes (1.5 ml), buffer G2, proteinase K and carrier RNA. All buffers and reagents can be stored at room temperature (15″25°C). Each kit is sufficient for 48 samples.The reagent cartridge contains all required reagents for DNA purification from a single sample. Each well of the cartridge contains a specific reagent, such as magnetic particles, lysis buffer, wash buffer, or elution buffer .
18.104.22.168 Principle and ProcedureMagnetic-particle method has the speed and efficiency of silica-based DNA purification characteristic as well as the convenient handling of magnetic particles (Figure 10). DNA is separated from lysates in the presence of a chaotropic salt by its binding to the silica surface of the particles. Due to the use of a magnet, the particles are removed from the lysates. The DNA is then well washed and eluted in either water or TE buffer. Elution volumes of 40 јl (EZ1 Advanced XL only), 50 јl, 100 јl, or 200 јl can be chosen.There are many different pre-treatment protocols optimized for each specific sample type, however they share main points including the following:1- The interested sample is placed in a 2 ml sample tube.2- A specified volume of proteinase K (table 5) is added to the sample followed by 10 seconds vortexing.3- Incubation at 56°C for 15 minutes to overnight in a thermomixer shaking at a maximum 900 rpm.4- EZ1 instrument set up and loading the sample tubes along with the required components of EZ1 DNA Investigator kit.5- Selecting the appropriate DNA purification protocol and run.6- UV decontamination. There are three DNA purification protocols. The first one is the standard protocol called Trace and designed to isolate total DNA from pre-treated samples as shown in table 5. In this protocol, when the sample is a solid material, like swabs and fabrics, it should be removed from the solution and centrifuged before processing DNA purification through EZ1 instrument. The second protocol which is useful for solid material such as swabs, fabrics, blood discs or cigarette butts, is called Tip Dance Protocol. In this protocol, the filter-tip moves back-and-forth relative to the worktable during pipetting and there is no need for prior centrifugation to remove solid materials. The last protocol for DNA purification available in EZ1 instruments is the Large-Volume Protocol. Here, up to 500 јl starting volumes can be processed and this activates DNA purification from dilute samples with low DNA concentrations and from samples that require larger volumes for complete lysis. Although starting larger volumes are processed, the elution yields more concentrated DNA ”.After the run is completed, the EZ1 instrument recommends UV decontamination for the worktable ,.The EZ1 DNA Investigator Kit contains Proteinase K, which is the enzyme used for lysis. It is a recombinant protein expressed in Pichia pastoris and is appropriate for short digestion periods. It characterizes with a great specific activity and stays stable over a wide range of temperatures and pH values, with substantially better activity at higher temperatures . 1.7 Separation of PCR Products by Capillary ElectrophoresisCapillary electrophoresis (CE) is a process used to separate amplified DNA fragments by size. A submission of high voltage charge to the sample forces the negatively charged fragments to move into the capillaries. The extension products are separated by size based on their total charge. There are many factors which can affect electrophoretic movement of the sample: the buffer type, concentration, pH; the run temperature; the amount of voltage applied; and the type of polymer used. Before reaching the positive electrode, the fluorescently labelled DNA fragments, separated by size, move across the path of a laser beam. Then, a laser beam causes the dyes attached to the fragments to fluoresce and a diffraction system separates the dye signals. Finally, the fluorescence is detected by a CCD camera (Figure 13). When the dyes are exposed to the laser, each dye emits light at a different wavelength, so the colors of the loci are detected and displayed on an electropherogram because the fluorescence signal is converted into digital data .1.8 ObjectivesThe main objectives of this study are the following:1- To study the indel polymorphisms among the UAE population.2- To study the forensic parameters of 30 indels in the UAE population.3- To evaluate the Investigator® DIPplex kit in forensic applications in the UAE population.