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GCT and OGTT were performed on the pregnant women. From each patient, 5?mL of venous blood was collected; 3?mL of the serum sample was used for the biochemical analysis Alisertib price to confirm the disease, and 2?mL of the EDTA sample was used for the molecular analysis. Genomic DNA was extracted from peripheral blood leukocytes using the salting-out technique as previously described (Movva et al., 2007). The DNA samples were stored at ??80?��C. The genotyping was performed at the Department of Genetics and Molecular Medicine (NABL accreditation laboratory), Kamineni Hospitals, Hyderabad, India for the rs2975760 polymorphism in intron 3 of CAPN10 by Polymerase Chain Reaction�CRestriction Fragment Length Polymorphism (PCR�CRFLP) followed by agarose gel electrophoresis on the extracted DNA using a pair of oligonucleotide primers: (forward sequence) 5��-GATGTGGGCATCCATAGCTT-��3 and (reverse sequence) 5��-TGATCCCATGGTCTGTAGCA-3��. The primers were synthesized by Bioserve Biotechnology (Hyderabad, India) for PCR analysis. The DNA was denatured at 95?��C for 5?min, amplified by 35?cycles see more of 95?��C for 30?s, 58?��C for 30?s, 72?��C for 45?s, and the final extension at 72?��C for 5?min. Initially, Fnu4HI (GC��NGC) (Thermo Scientific, USA) enzyme was tested with �� DNA to confirm the restriction enzyme and the PCR products were digested for 4?h with Fnu4HI (GC��NGC) (Thermo Scientific, USA) at 37?��C (2.5?��L of distilled water with 10?units of enzyme per 15?��L PCR product and 2?��L of buffer in a final volume of 20?��L). The digested PCR products (rs2975760) were electrophoresed Bumetanide on 3.5% agarose gel containing ethidium bromide. The data are expressed as the mean?��?SD. Student's t-test for unpaired data was used. The allele and genotype frequencies were determined by allele counting, and the chi-square (��2) test was used to compare the observed versus the expected outcomes. The qualitative data were compared by the ��2 test. The allele frequencies were estimated by the gene counting method, and the ��2 test was used to identify departures from Hardy�CWeinberg equilibrium (HWE). The statistical significance was examined by two-sided tests, and the statistical analyses were performed with SPSS version 19.0 software. A p value of