Fastdigest hpaii

Buccal cells were harvested from the inner cheek of each subject to provide DNA for genetic testing. The DNA was extracted from buccal swabs using a QIAamp DNA Mini Kit. The procedure employed a polymerase chain reaction (PCR)–based protocol followed by restriction endonuclease digestion to identify the 5-HTTLPR that are located in the promoter region of the serotonin transporter gene (SLC6A4) and rs25531 variants: S, LA, and LG. Forward primer: 5′-TCCTCCGCTTTGGCGCCTCTTCC-3′ and reverse primer: 5′-TGGGGGTTGCAGGGGAGATCCT-3′ (10 μM each) were used for 50 μL PCR containing approximately 25 ng DNA, 25 μL Taq DNA Polymerase 2× Master Mix Red (Ampliqon) and ddH2O, with an initial 5-min denaturation step at 95°C followed by 35 PCR cycles of 95°C (30 s), 65°C (40 s), and 72°C (30 s) and a final extension step of 5 min at 72°C. To distinguish the A/G single-nucleotide polymorphism of the rs25531, we extracted 10 μL of the PCR product for digestion by FastDigest HpaII (Thermo, FD0514), an isoschizomer of MspI, a total reaction of 20 μL. These were loaded side by side on 2.5–3.0% agarose gel.

One μg of genomic DNA isolated from peripheral blood was digested with FastDigest HpaII in a 20 µl reaction volume using 1 µl of restriction enzyme according to standard protocol (Thermo Fisher Scientific, Waltham, MA). Amplification of the androgen receptor (AR) microsatellites was carried out in a 20 µl reaction containing 0.1 mM of dNTP, 250 nM of each primer, 1.25 mM of MgCl₂, 2 µl of buffer, 1 U of Taq polymerase, and 50 ng of DNA or 2 µl of digested DNA, with primers and cycling conditions as previously described^{25 (link)}. Amplification of the retinitis pigmentosa 2 (RP2) gene microsatellites was performed as previously described^{26 (link)} with an input of 50 ng of DNA or 2 µl of digestion product. 5′ primers were marked with (6)-Carboxyfluorescein protein (/56-FAM; Supplementary Table S2). Genotyping was performed through a fragment length analysis (FLA), as described in the Supplemental Data 1, and analyzed with the GeneMapper™ software (Thermo Fisher Scientific, Waltham, MA). Primer sequences are stated in Supplementary Table S2.