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© 1993 Oxford University Press

RESEARCH-ARTICLE

Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously amplified ZFX and ZFY sequences

Samuel S. Chong, Kristlelfur Kristjansson, Juan Cota, Alan H. Handyslde1 and Mark R. Hughes*

Institute for Molecular Genetics and Center for Reproductive Medicine, Baylor College of Mediane and The Methodist Hospital, Houston, TX 77030. USA 1Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital London W12 ONN, UK

* To whom correspondence should be addressed

Received February 1, 1993; Revised May 3, 1993; Accepted May 3, 1993

In vitro fertilization (IVF), blastomere biopsy of the 6–8 cell embryo, and single cell DNA diagnosis allows couples at risk of transmitting an X-linked or autosomal disease to start a pregnancy knowing their child will not be affected. We present a quick and reliable nested PCR strategy for sex determination at the single cell level by simultaneous amplification and subsequent restriction fragment analysis of the homologous but non-allellc ZFX and ZFY genes present on the X and Y chromosomes respectively. Amplified ZFX and ZFY sequences are of equal size and produce distinguishable Haelll digestion products. In a randomized, blinded study of 194 Individually isolated lymphoblasts, amnlocytes, chorion villus cells, and blastomeres, 191 amplified successfully (98.4% sensitivity). None of the sample blanks showed any PCR product, all 90 of the karyotyplcally XY cells were correctly genotyped as ZFX/ZFY, all 83 of the 84 XX cells that amplified were correctly genotyped as ZFX only, and analyses of all same-embryo blastomeres were completely concordant (100% specificity). This strategy avoids a source of misdlagnosis observed in methods which detect only Y-specific sequences, where amplification failure In an XY cell results in an erroneous XX diagnosis. This rapid (6 hr) and simple method of analysis, when applied to preimplantation embryo diagnosis, allows the avoidance of offspring affected with an X-linked recessive disorder by transferring only female embryos for Implantation and ensuing pregnancy.


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