An accurate gender determination assay from a small amount of dna specimen as an aid for non-invasive prenatal diagnosis

For clinical application of genetic diagnosis from blastomeres, exfoliative fetal cells of the uterine cervix, or circulating fetal cells in the maternal blood, we have developed an accurate multiplex polymerase chain reaction (PCR) assay for gender determination from a small amount of DNA specimen. This assay co-amplifies X-and Y-specific repetitive sequences (DXZ1/DYZ1), so that 2 bands (DXZ1 and DYZ1 positive) implies male; single band (only DXZ1) means female; no band suggests amplification failure or sampling error. Each band was confirmed to be specific by the direct PCR products sequencing method. To evaluate accuracy and efficiency of this assay in single cell, we sampled a single amniocyte (n - 50) with a micromanipulator under an inverted microscope, extracted DNA from it, and analyzed. Sex determination by this PCR method was compared with that by karyotyping. The amplification success rate was 98% (49/50) and misdiagnosis was not observed. This newly developed assay appears efficient and accurate, thus may be applicable to the above mentioned clinical settings.