| Project/Area Number |
06671683
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Keio University |
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Principal Investigator |
KOBAYASHI Toshimi Keio Univ..School of Medicine, associate professor, 医学部, 助教授 (30051460)
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| Co-Investigator(Kenkyū-buntansha) |
HASHIBA Tsuyoshi Keio Univ., School of Medicine, assistant, 医学部, 助手 (60245553)
ASADA Hironori Keio Univ., School of Medicine, assistant, 医学部, 助手 (60231883)
KUJI Naoaki Keio Univ., School of Medicine, assistant, 医学部, 助手 (80169987)
SUEOKA Kou Keio Univ., School of Medicine, assistant, 医学部, 助手 (90162833)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | X・Y sperm preseparation / SRY gene / PCR / FISH / DXZ-1 / DYZ-1 / alpha-Satellite repeat sequence / Percoll遠心分離法 / α-satellite repeat sequence |
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| Research Abstract |
More than 20 candidates have undergone preimplantation gender determination by polymerase chain reaction (PCR), but a more accurate, rapid and simple method has been needed for general clinical application. We have developed a new multiplex PCR assay for gender determination, those employing co-amplification of the X and Y chromosome specific repeated sequences. We have evaluated the efficiency and accuracy of this assay and measered the time needed for this assay. The most optimum condition for the PCR assay was determined and the results of sexing by the assay in 50 amniotic fluid samples were compared with that of sexing by culture and karyotyping. Fifty single 46XX amniocytes and 50 single 46XY amniocytes were analyzed by this assay. The efficiency and accuracy of the assay in single cell was evaluated. Single amniocytes were isolated under an inverted microscope equipped with micromanipulators. The X1-1, X1-2 primer set was derived from the pericentric region of the X chromosome (DXZ-1). The Y1-1, Y1-2 primer set was derived from the long arm of the Y chromosome (DYZ-1). The X-and Y-derived amplified fragment ware 308 and 154 base pairs, respectively. Using this multilex PCR assay, we could diagnose fetal sex accurately in 50 consecutive amniotic fluid samples (46XX : 17 cases, 46XY : 31 cases, 47XY,+18 : 1 case, 46XX,t (1 : 3 : 16) : 1 case). We analyzed 50 single 46XX amniocytes and 50 single 46XY amniocytes. Ninety-nine out of 100 amniocyte samples (99%) were accurately diagnosed and only one sample was not amplified. No contamination was observed. The time needed for this assay was 2 hours. This multiplex PCR assay for gender determination in single cell is effeicient and accurate and can be used for preimplantation diagnosis. This assay needs only 2 hours and will not disturb implantation rate.
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