Patients. Of the 40 Turner syndrome patients we studied, 36 were hospitalized or out patients of the Department for Endocrinology and Genetics at the University Pediatric Clinic in Skopje, Republic of Macedonia, and four were out-patients of the University Clinic for Adult Endocrinology in Skopje. Inclusion criteria were a typical phenotype of Turner syndrome and the result of cytogenetic analysis. The mean age was 16 years.

      Cytogenetic Analyses. Peripheral blood lymphocytes were examined by G banding (banding resolution of 400 bands, average number of observed metaphase preparations, 50).

      DNA Extraction. Genomic DNA was extracted from 2-6 mL ethylenediamine tetraacetate-containing blood using a standard phenol-chloroform procedure from peripheral blood leucocytes [23,24] and from one formalin- treated and paraphine-embedded ovarian sample [25] extirpated due to endometrial malignancy.

      Polymerase Chain Reaction Amplification. Two sets of oligonucleotide primers were used: XES7/XES2 and SRY 1F/SRY 2R. XES7 5'-GAC AAT GCA ATC ATA TGC TTC TGC-3'/XES2 5'-CTG TAG CGG TCC CGT TGT GCG GTG-3' amplify a 609 bp fragment that spans almost the entire open reading frame (ORF) of the SRY gene [4,5,26,27] (Figure 1). SRY 1F 5'-CAG TGT GAA ACG GGA GAA AAC AGT-3'/SRY 2R 5'-CTT CCG ACG AGG TCG ATA CTT ATA-3' amplify a 270 bp fragment (518-788 bp) that mainly encompasses the HMG-box domain, an evolutionary highly conserved motif that codes for a protein with DNA-binding characteristics [27] (Figure 1). The primers were synthesized in Sigma Genosys (Sigma-Aldrich Corporation, St. Louis, MO, USA). Amplification of a 165 bp fragment of the angio­tensinogen gene or a 458 bp fragment of the CFTR gene were used as controls. The sequences of the primer pairs used to amplify the internal controls were: F 5'-CAG GGT GCT GTC CAC ACT GGA CCC C-3'/R 5'-CCG TTT GTG CAG GGC CTG GCT CTC T-3' for the first fragment, and F 5'-TCA CAT ATG GTA TGA CCC TC-3'/R 5'-TTG TAC CAG CTC ACT ACC TA-3' for the second fragment.

      The PCR amplification was performed in a final volume of 50 mL, the reaction mixture consisting of 300-500 ng genomic DNA, 50 pmol of each specific primer and 30 pmol of the control primers, 1.5 U AmpliTaq Gold polymerase (Applied BioSystems, Foster City, CA, USA), 2 mM MgCl₂, 200 mM 4 ´ dNTP, commercial PCR buffer. The amplification was carried out with a DNA thermal cycler (Perkin Elmer 480 version 2; Perkin Elmer Corporation, Waltham, MA, USA); 33 cycles with different programs for each primer set (XES7/XES2: 94°C, 10 min., 94°C, 45 seconds, 60°C, 1 min., 72°C, 2 min.; SRY 1F/ SRY 2R: 94°C, 10 min., 94°C, 45 seconds, 58°C, 1 min., 72°C, 2 min.) (amplification conditions were based on a method previously reported [27] and self- adjusted temperatures).          

       Analyses of the Polymerase Chain Reaction Amplified Products. All PCR products (10 mL) were electro­phoresed on a 2% agarose gel in 1 ´ TBE buffer stained by ethidium bromide and visualized under UV light. Several precautions were taken to avoid false-positive results [7]. All laboratory procedures were performed by a female operator, thus excluding the possibility for sample contamination with male cells. Pre- and post-PCR work spaces were strictly separated so that carry-over of amplified DNA sequences to new PCR reactions were prevented. Each PCR reaction contained one normal female and one template-free sample for early detection of contamination. Each PCR reaction included one normal male sample as a positive control.