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- Title
Development of preimplantation genetic diagnosis (PGD) for insertion and point mutation of Duchenne muscular dystrophy (DMD).
- Authors
Hiroto, TAJIMA; Kou, SUEOKA; Shinichi, TSUCHIYA; Taro, MAEDA; Akira, NAKABAYASHI; Kenji, SATO; Tsuyoshi, HASHIBA; Hironori, ASADA; Sokichi, IWATA; Naoaki, KUJI; Yasunori, YOSHIMURA; Shiro, NOZAWA
- Abstract
Objective We have developed accurate PGD protocols of single gene deletion using multiplex nested PCR instead of gender determination for DMD. However, other mutations of single base insertion or point mutation need direct sequencing following nested PCR. The diagnostic accuracy was examined for PGD. Methods Nested PCR from single cell was performed from blood samples of normal men, women, and DMD patients (single base insertion/point mutation). All specimens were used under the informed consent. The following experiments were performed; (1) most sensitive conditions for nested PCR to exon45 were investigated (2) diagnostic accuracy was compared between gender determination using DYZ-1 and single gene amplification (exon45), and (3) diagnostic efficiency for DNA sequencing was examined using specimens of DMD patients. Results (1) The accuracy was 90% when the nested PCR condition was 30 cycles each for lst/2nd PCR, and annealed at 64/ 62°C for 60 sec. (2) While DYZ-1 with thousands of repeated copies was amplified in all specimens from single cell, exon45 was amplified in 90% of cases. (3) The sequencing was successfully analysed in all specimens when nested PCR was successfully amplified.
- Subjects
PREIMPLANTATION genetic diagnosis; DIAGNOSIS of Duchenne muscular dystrophy
- Publication
Journal of Obstetrics & Gynaecology Research, 2003, Vol 29, Issue 3, p216
- ISSN
1341-8076
- Publication type
Article