2.3. Detection of FecXI, FecXH, FecXG, and FecXB Mutation Sites of the BMP15 Gene

With reference to previous research [6,8,10], four pairs of primers were designed for FecXI, FecXH, FecXG, and FecXB to detect the BMP15 gene. These primers are listed in Table 1.

Primers of FecXI, FecXH, FecXG, and FecXB mutation sites of the Bone Morphogenetic Protein 15 (BMP15) gene.

The PCR reaction volume of 20 µL contained Taq DNA polymerase 1.5 U, 10 × buffer 2 µL, MgCl2 (25 mmol/L) 1.2 µL, dNTP (2.5 mmol/L) 2 µL, genomic DNA ~50 ng, as well as upstream and downstream primers (each 10 pM). Water was added at 20 µL.

The above sites were detected using the Touch-Down PCR reaction program. The reaction conditions were as follows: (1) 95 °C pre-denaturation for 5 min; (2) Annealing temperature for PCR was performed by gradient PCR which 95 °C denaturation for 30 s, 65 °C–51 °C (65 °C, 63 °C, 61 °C, 59 °C, 57 °C, 55 °C, 53 °C, 51 °C) annealing for 40 s, 72 °C extension for 30 s (each annealing temperature of 65 °C–53 °C for two cycles respectively and 51 °C for 19 cycles); (3) finally, extension at 72 °C for 5 min. The product was detected by 2% agarose gel electrophoresis.

Restriction enzyme digestion of the PCR product BMP15 gene FecXH mutation (CT) was amplified with the Spe I enzyme. The BMP15 gene FecXI mutation (TA) was amplified via Xba I endonuclease digestion. The BMP15 gene FecXG mutation amplification product was digested with the Hinf I enzyme. The FecXB mutation was amplified and digested with Dde I endonuclease. The digestion reaction system used 4 μL PCR product, 1 μL 10× buffer, and 5 U endonuclease filled to 10 μL with double-distilled water. The digestion used a water bath at 37 °C for 12 h. The digested product was detected by 3% agarose electrophoresis.