# Genetic Variants in Patients with Premature Ovarian Insufficiency in a Mexican Cohort
## 2 Materials and Methods
### Genetic Screening and Genotyping  
Mutations were identified via PCR-single-strand conformation polymorphism (SSCP) analysis. All coding exons of the _WT1_ (NM_024426.6; Region 11: 32409321-32457176), _NROB1_ (NM_000475.5; Region X: 30322323-30327715), _NR5A1_ (NM_004959.5; Region 9: 127243516-127269709), _LHK9_ (NM_020204.3; Region 1: 197881037-197904608), _ZFP29_ (NM_001136273.2; Region X: 152683780-152687087), _ZNF275_ (NM_001080485.4; Region X: 152599613-152625568), _INSL3_ (NM_001265587.2; Region 19: 17927321-17932383), and _NRIP1_ (NM_003489.4; Region 21: 16333556-16437321) genes, including their intron boundaries, were individually amplified from 300 ng/\(\upmu\)L of gDNA via polymerase chain reaction (PCR) using (\(\upalpha\)-\({}^{32}\)P)-dCTP-specific activity (3000 Ci/mmol; PerkinElmer, Boston, MA, USA) and GoTaq Fexi DNA Polymerase, according to the supplier's guidelines (Promega, Madison, WI, USA) in a final reaction of 20 \(\upmu\)L. The PCR reactions contained 25 \(\upmu\)M of dNTP and 20 \(\upmu\)M of each sense and antisense oligonucleotide (Supplementary Tables S1-S8). For all exons, the MgCl\({}_{2}\) concentrations and melting temperatures were calculated experimentally. The PCR conditions were: 1 cycle at 94 \({}^{\circ}\)C for 3 min; 30 cycles at 94 \({}^{\circ}\)C for 30 s, 57-65 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 30 s; and 1 final extension at 72 \({}^{\circ}\)C for 3 min. The (\(\upalpha\)-\({}^{32}\)P)-dCTP-PCR reactions were visually assessed on 1% agarose gels containing 0.5 \(\upmu\)L/100 mL ethidium bromide run at 100 V for 1 h. A total of 77 agarose gels were required to analyze the eight candidate genes. Exonic amplifications were observed using a UV transilluminator (Molecular Imager Gel Doc XR System, BioRad Laboratories, Hercules, CA, USA). Amplicon size was determined by comparison to a 100 bp molecular weight marker.  
To detect genetic variants or exonic mutations, each exon amplified via (\(\upalpha\)-\({}^{32}\)P)-dCTP-PCR was analyzed by electrophoresis in four denaturing polyacrylamide gels using SSCP. After each (\(\upalpha\)-\({}^{32}\)P)-dCTP-PCR amplification, 1 mL of (\(\upalpha\)-\({}^{32}\)P)-dCTP-PCR reaction was mixed with 14 mL of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue). The samples were denatured at 95 \({}^{\circ}\)C for 5 min and cooled for 5 min. One microliter of each sample was loaded onto four polyacrylamide gels. Two 8% polyacrylamide gels with and without glycerol (7 mL) were prepared using 14 mL of TBE 5X, 18.66 mL of acrylamide-\(N\),\(N^{\prime}\)-methylenebisacrylamide (29:1), 0.48 mL of 10% ammonium persulfate (APS), and 25 \(\upmu\)L of tetramethylethylenediamine (TEMED) in a final volume of 70 mL with H\({}_{2}\)O, and two 5.4% polyacrylamide gels with and without glycerol (7 mL) were prepared using 14 mL of TBE 5X, 12.6 mL of acrylamide-\(N\),\(N^{\prime}\)-methylenebisacrylamide (29:1), 0.48 mL of 10% APS, and 24.5 \(\upmu\)L of TEMED in a final volume of 70 mL with H\({}_{2}\)O. Electrophoresis was carried out at 200-250 volts for 18 h at room temperature, after which the polyacrylamide gels were dried for 1 h at 75 \({}^{\circ}\)C and exposed to Imaging Screen-K screens for 3 h. A total of 308 polyacrylamide gels were required to screen the eight candidate genes. The results were analyzed using the Personal Molecular Imager System (Bio-Rad Laboratories, Hercules, CA, USA). Exons with aberrant migration patterns on the SSCP gels were amplified by PCR without (\(\upalpha\)-\({}^{32}\)P)-dCTP and purified using electroelution (Spectra/Por 1 Dialysis Membrane, MWCO: 6-8 kD, Spectrum Laboratories, Inc, Rancho Dominguez, CA, USA) and Amicon Ultra-4, Ultracel-10 K centrifugal filter devices at 1500\(\times\)\(g\) for 20 min (Merck Millipore Ltd., Carrigtwohill, Co., Cork, Ireland). The allelic and genotypic frequencies of 100 unrelated, healthy subjects were determined using PCR-SSCP assays.