amplicon, homo,T2DM,CARD9
Genomic DNA extraction from blood collected in EDTA tubes was done automatically using magnetic beads (MagNA Pure 96 (Roche, Germany)). Gentic amplification and sequencing was performed at the Center for Genetics and Inherited Diseases, Taibah University, Medina, Saudi Arabia. Targeted genotyping of specific primers of CARD9 exon 2 and 3 (Homo sapiens caspase recruitment domain family member 9 (CARD9), RefSeqGene (LRG_178) on chromosome 9, NCBI Reference Sequence: NG_021197.1) were used to amplify single nucleotide polymorphisms (SNPs) (Primers Forward GTCTGAGAAGGAGTGGGAGC, reverse GCTGTGGCAGGAGCTCAGG) which amplified a 192bp product. PCR amplification was conducted using the GoTaq Green Master Mix (2X) (Promega, USA) in a total reaction volume of 25 L for diagnostic screening and scaled to 35 L for downstream sequencing applications. Each reaction mixture contained:12.5 L of 2X GoTaq Green Master Mix (final concentration 1X). Upstream and downstream primers (10 M stock) at a final concentration ranging from 0.1 to 1.0 M, 2 L of DNA template and Nuclease-free water to reach the final reaction volume. the reactions were performed using a Biometra TOne thermal cycler (Analytik Jena, Germany). The thermocycling conditions consisted of an initial denaturation at 95C for 5 minutes, followed by 45 cycles of(Denaturation: 95C for 30 seconds, annealing: Optimized between 45C and 68C for 45 seconds, extension: 72C for 45 seconds, final extension step was performed at 72C for 5 minutes, followed by a cooling hold at 4C). Amplification success and product size were verified via agarose gel electrophoresis. A 1% (w/v) agarose gel was prepared by dissolving 1g of agarose in 100mL of electrophoresis buffer (TBE). To allow for nucleic acid visualization, 3 L of ethidium bromide was added to the gel mixture prior to casting. Electrophoresis was conducted at 100V voltage for 45 minutes and DNA bands were visualized under UV transillumination (Geldoc biorad). After PCR purification Sanger sequencing was used as a genotyping method. Post-PCR amplicons were enzymatically purified using the ExoSAP-IT kit to remove excess primers and dNTPs, ensuring a clean template for downstream processing. These purified products were then subjected to cycle sequencing utilizing the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Following cycle sequencing, the fragments were purified through ethanol precipitation and subsequently resolved by capillary electrophoresis on an Applied Biosystems 3500 Genetic Analyzer