Base reagent preparation

All critical reagents for the mitochondrial assay are independently tested for performance and template contamination prior to kitting. One of the most important critical reagents is the primer pairs and subsequent triplexes that are used in the mitochondrial assay. This process is thoroughly document with SOPs for making the primer pairs and triplexes as well as test methods for quality control (QC) of the material at critical steps. The first step in making primer pairs is the resuspension of individual primers to 750 μM based on vendor provide quantification and then quantification by UV spectrometry. Accurate quantification is necessary to ensure that the primer pairs are mixed together at the same concentration. The primers are then diluted to a uniform concentration of 500 μM based on the UV quantification. Thus, when the primer pairs are mixed one –to –one, the resulting solution is 500 μM total (250 μM each primer). According to an appropriate test method, the primer pairs are tested for performance and contamination using our current positive control of Seracare blood sample SC35495. This step is designed to make sure that the intra-strand ratios of the PCR products are within the defined tolerances as well as looking for performance and contamination. In several instances, primer pairs were found that were contaminated or that did perform PCR at all. These primer pairs were destroyed and not used in the next step. Once all twenty-four primer pairs pass QC, the primer pairs are then combined into their appropriate triplexes and undergo performance and contamination testing again. In addition, the product balance in each triplex is also examined using the positive control. To test the performance of the triplexes, PCR reactions are performed using 25 pg, 100 pg, and 500 pg of input DNA. Full correct profiles must be obtained for all three dilutions in order for the triplexes to pass QC. In addition, three replicate PCR reactions with no template added must show no contamination in order for the triplexes to pass QC.

The next critical reagent is the dNTP mix used for PCR. We make our own dNTP mix because we use a carbon 13-enriched dGTP in the mitochondrial assay. Since the

dGTP is enriched in carbon 13, the percent enrichment needs to be determined prior to its use in kitting plates. In addition, the dNTP mix must past performance and contamination criteria. Using previously passed triplexes, three replicate no-template­ added PCR reactions are used to test for contamination. To test the performance of the dNTP mix, PCR reactions are performed using 25 pg, 100 pg, and 500 pg of input DNA. These replicate reactions are also used to determine the percent enrichment of the carbon 13-enriched dGTP.

A third critical reagent is the PCR master mix base used in kitting the plates. The PCR master base contains all the reagents necessary for PCR except for the dNTPs, the triplexes and the Taq enzyme. This reagent is tested for contamination and performance in the same manner as the triplexes and the dNTP mix using previously passed lots of triplexes and dNTP mix. In addition, the PCR product balance within a triplex is examined. Various lots of PCR master mix base have been shown to influence the product ratios and thus, the product ratios must be within acceptable tolerances in order for the PCR master mix base to pass QC.

Each lot of Taq enzyme is also tested for contamination and performance. To date, the Taq enzyme has always meet specifications. The 96-well PCR plates that are used for kitting the reagents are UV treated for 10 minutes in a UV cross linker oven to minimize contamination in the plates. The plates are UV treated prior to the kitting and sealed with a removable heat seal that allows the plates to be stored contamination-free.

Once all of these reagents pass QC, kitted plates are made using a closed system to minimize the possibility of outside contamination. Typical kitting runs produce 400 plates. In process QC is performed on the first and every one hundredth plate to ensure that plate volumes are in acceptable range. Prior to dispensing reagents, the weight of the empty plate is recorded. After dispensing the reagents, the weight of the plate is recorded again. The difference between the two weights is divided by 96 and multiplied by 1000 to give an average volume per well per plate. If the average volume per well does not meet specifications, the dispenser is adjusted and tested as above until the

volume is within the acceptable range. These same plates that are used for in-process QC are used for functional and contamination QC. For each first and one hundredth plate, performance and contamination is evaluated by putting primer dilution buffer in columns 1, 2, 5, 6, 9 and 11 as a no-template-added control as well as 500 pg of the positive control in columns 3, 4, 7, 8, 10 and 12. These plates indicate how reproducible the PCR is across the plate as well as test for contamination in the kitting process. Full correct profiles must be obtained for all positive controls and the negatives must be free of contamination in order for the plates to pass. In addition the last plate of the batch is used to test for sensitivity. The sensitivity is tested by using 1 pg, 4 pg, 10 pg, 25 pg, 100 pg and 500 pg inputs of the positive control DNA. A full correct profile must be obtained down to 25 pg of input DNA in order for the plates to pass QC. Typically, full correct profiles are observed between 4 pg and 10 pg of input DNA.

The above processes are all documented and the methods are in the process of being transferred to our manufacturing facilities. Once the formulation for the STR assay has been finalized, the same processes employed to quality control the reagents and kitted plates for the mitochondrial assay will be applied to this assay.