Abi 3500 genetic analyzer

In total, 929 negative control samples were collected from six laboratories (LAB_a–f) between 2019 and 2022. The amplification kits used included the AGCU EX22 kit (Applied ScienTech, Wuxi, China), PowerPlex 21 kit (Promega, Madison, WI, USA), and VeriFiler™ Plus kit (Thermo Fisher Scientific, Waltham, MA, USA). Amplified products were analyzed using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Additionally, the experiments on low-template DNA samples were conducted by seven laboratories (LAB_a–e, g and h) using the VeriFiler™ Plus kit. All experiments were consistently performed by the same experimenter. Female control DNA 9947A (OriGene, Rockville, MD, USA) was diluted to three concentrations: 31.25 pg/µL, 15.625 pg/µL, and 7.8125 pg/µL. Each PCR reaction used 1 µL of DNA, with a total volume of 10 µL, following the routine protocol for the cases. Three PCR replicates were conducted for each concentration of 9947A and negative control for 27, 29, and 31 cycles. The amplified products were separated via capillary electrophoresis using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), with three replicates for each amplified product. The research protocol was reviewed and approved by the Ethics Committee at the Institute of Forensic Medicine, Sichuan University (No. KS2022770) (4 March 2022).

Haplotype analysis was performed using four microsatellite markers (from telomeric to centromeric, D9S169, R14866, D9S191, D9S263) located at 9p21.2 around the C9ORF72 gene. Microsatellite typing was performed by fluorescent PCR and the fragments’ lengths were stablished by capillary electrophoresis on an ABI3500 Genetic Analyzer using GeneScan^TM 600 LIZ^TM Size Standard (ThermoFisher Scientific).

PCR followed by fragment analysis by capillary electrophoresis (ABI 3500 Genetic Analyzer) was performed for FLT3-ITD testing. The PCR was performed starting from 50 ng of gDNA in a total reaction volume of 25 μL, containing the following: 1U of Taq DNA Polymerase, 0.5 μL dNTP mix (10 mM), 1× PCR Buffer, 2 mM MgCl₂, and 0.5 μM of each primer. The primers for ITD detection were fluorescently labeled: 5′-HEX-GCAATTTADGTATGAAAGCCAGC-3′ (forward), and 5′-FAM-CTTTCAGCATTTTGACGGCAACC-3′ (reverse) [33 (link)]. The cycling conditions were as follows: denaturation for 3 min at 94 °C; 40 cycles of 45 s at 94 °C, 60 s at 61°C, 90 s at 72 °C; and final extension for 15 min at 72 °C. The amplification products were denatured with Hi-Di Formamide and migrated on ABI 3500 Genetic Analyzer (Thermo Fisher Scientific), using the GeneScan 600 LIZ Standard (Thermo Fisher Scientific) for size evaluation. The data were analyzed with the GeneMapper Software v5 (Thermo Fisher Scientific); the amplicons larger than the expected wild type (330 bp) were interpreted as positive for the ITD variant.