Hpaii

DNA was extracted from female patients with DDCS according to the above procedure, and normal female blood sample DNA was used as a control. The digestion reaction system (final volume = 20 μl) included 10 μl DNA, 1 μl HpaII (Takara, Dalian, China), 2 μl 10× loading buffer, and 7 μl dH₂O. Distilled water instead of enzyme was used in the nondigested system as a control. The reaction conditions were 37 °C digestion for 3 hours and then incubation at 85 °C for 15 minutes to stop the reaction. This was followed by PCR using a 50 μl reaction system including 10 μl 5× PrimeSTAR GXL Buffer, 4 μl dNTP mix, 1 μl PrimeSTAR GXL DNA polymerase (Takara, Dalian, China), 1 μl each primer (AR) (Sangon Biotech, Shanghai, China), 28 µl sterile water, and 5 μl digested or nondigested DNA. The sequences of the primers were 5′-CTACCGAGGAGCTTTCCAGAAT-3′ (forward primer, labeled with FAM green fluorescence on the 5′ end) and 5′-CGATGGGCTTGGGGAGAACCAT-3′ (reverse primer). The mixture underwent predenaturation at 95 °C for 5 minutes, followed by 30 cycles at 98 °C for 10 s and 68 °C for 30 seconds. The PCR product was used for capillary electrophoresis on an ABI 3730 sequencer (Applied Biosystems), and the data were analyzed by GeneMapper 4.0 Software (Applied Biosystems).

Bovine serum albumin (BSA; essentially fatty acid and globulin-free), Trizma base, 2-mercaptoethanol, 3-aminopropyltriethoxysilane and Brij-35 were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); 3,3′-Diaminobenzidine-4HCl (DAB) was purchased from Chemical Dojin Co., Ltd. (Kumamoto, Japan). Biotin-16-dUTP, digoxigenin-11-dUTP, Rhodamine anti-Dig and terminal deoxynucleotidyltransferase (TdT) were from Roche Diagnostics (Mannheim, Germany). Dideoxy ATP (ddATP) and dideoxy TTP (ddTTP) were from Jena Bioscience (Jena, Germany). HpaII and MspI were purchased from Takara Bio, Inc. (Otsu, Japan). DAPI was obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Permount was from Thermo Fisher Scientific, Inc. All other reagents used in the present study were obtained from Wako Pure Chemicals Industries, Inc. (Osaka, Japan) and were of analytical grade.

We performed XCI analysis using FRAXA locus methylation assay as previously described (62 (link)). Briefly, genomic DNA from the subjects was digested with two methylation-sensitive enzymes, Hpa II and Hha I (Takara). The digested DNA was PCR-amplified with fluorescent-labeled primers targeting a triplet repeat of FRAXA gene. See table S6 for a list of primer sets. Fluorescent-labeled products were analyzed on an ABI PRISM 3500 Genetic Analyzer with GeneMapper Software version 4.0 (Applied Biosystems). X-inactivation ratios of less than or equal to 80:20 were considered to represent a random pattern, ratios greater than 80:20 were considered to represent a skewed pattern, and ratios greater than 90:10 were considered to represent a markedly skewed pattern. Fragment length along with an appropriate standard was determined using a 3730xl DNA Analyzer (Applied Biosystems) and analyzed with Peak Scanner Software 2 (Applied Biosystems).