Mutations of the androgen receptor gene in patients with complete androgen insensitivity

✍ Scribed by Sibylle Jakubiczka; Stefanie Nedel; Edmond A. Werder; Engelbert Schleiermacher; Ursel Theile; Gerhard Wolff; Peter Wieacker

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 185 KB
• Volume: 9
• Category: Article
• ISSN: 1059-7794
• DOI: 10.1002/(sici)1098-1004(1997)9:1<57::aid-humu10>3.0.co;2-o

✦ Synopsis

Communicated by Stylianos E. Antonarakis

fied by polymerase chain reaction (PCR) ace cording to Saiki et al. (1988) using the intronic primers of Lubahn et al. (1989). Routinely, 32 cycles were run in an Intelligent Heating Block (Biometra) after a first denaturation step for 5 min at 93°C, 60 s at 93°C, 60 s at the annealing temperature (exon B: 62°C; exon C, D, F: 58°C; exon E: 68°C, exon G: 60°C, exon H: 50°C), 60 s at 72°C. A final elongation step for 10 min at 72°C was carried out.

Single-Strand Conformation Polymorphisms Analysis

For SSCP-analysis, the polymerase chain reaction (PCR) reaction consisted of 100 ng of DNA, 5 pM primer, 200 ãM of each dNTP, 0.25 ãCi [~-33 P]dATP and 1 U Taq-polymerase in 20-ãl suppliers buffer (Amersham). Amplification was performed under the same conditions as described above. Products from exon B, C, D, G, and H were restricted by HindIII, HhaI, DdeI, SphI, and MspI, respectively, in order to obtain fragments smaller than 300 bp. All products were run overnight at 10 W on 5% polyacrylamidgels (4°C) and on 5% polyacrylamidgels containing 10% glycerol (20°C).

DNA Sequencing

Sequencing of PCR products was carried out according to Dörk et al. (1991) by separating the double-stranded PCR-products by three ultrafiltration steps through centricon columns (Amicon); the