Haruo Takabayashi

Whole genome amplification from single cells in preimplantation genetic diagnosis and prenatal diagnosis.

The literature on whole genome amplification (WGA) techniques and their application to preimplantation genetic diagnosis (PGD) and prenatal diagnosis is reviewed. General polymerase chain reaction (PCR) fails to provide adequate information from limited cells in PGD and non-invasive prenatal diagnosis. Therefore several WGA techniques, such as primer extension preamplification (PEP) and degenerate oligonucleotide primed PCR (DOP-PCR), have been developed and successfully applied to clinical work during the past decade, especially in PGD and prenatal diagnosis. These techniques can provide ample amplification of genetic sequences from single cells for a series of subsequent PCR analyses such as restriction fragment length polymorphisms (RFLP) and comparative genomic hybridization (CGH), thus opening up a new area for prenatal diagnosis. However, several problems have been reported in the application of these techniques. The ideal WGA technique should have high yield, faithful representation of the original template, complete coverage of the genome, and simply performed procedure. In order to make good use of these techniques in future research and clinical work, it is undoubtedly necessary for an extensive understanding of the merits and pitfalls of these recently developed techniques.

Fetal DNA Diagnosis from Maternal Blood

Abstract: Development of a real‐time quantitative TaqMan PCR assay permits the high‐quality analysis of DNA or RNA. This study demonstrates the successful detection of single locus genes from single NRBCs, retrieved by micromanipulation of cells on a blood smear slide. Quantitative data in relation to the concentration of a single locus DNA amplified by PEP from a single NRBC are also provided. In summary, a PEP‐TaqMan system can potentially be a powerful tool for noninvasive fetal DNA diagnosis through the use of maternal blood.

398. Flow Cytometric Analysis on Nuclei Isolated from the Human Chorionic Tissues in Various Gestational States

異常妊娠および絨毛性疾患のflow cytometryによる病態解明を目的として, 生理的, 病的条件下のヒト絨毛組織細胞の相対的核DNA螢光量を測定し, その増殖動態を検討した.絨毛組織 (妊娠初期, 妊娠後期, 流産, 重症悪阻, 妊娠中毒症, 胞状奇胎) を採取し, クエン酸処理法により裸核化した.裸核化した検体を固定後, propidium iodideにて染色し, Cytofluorograf50Hにより相対的核DNA量を測定した.データは, Friedの解析法とTakahashiの解析法を折衷し藤川 (1984) の開発したcell cycle analysis program (Fujikawaの解析法) により解析した.その結果は, 以下のとおりである.1. 妊娠前期において, 妊娠後期と比較して, 増殖期細胞の顕著な増加がみられた.polyploidyやaneuploidyはみられなかった.2. 異常妊娠において, その増殖動態に正常群と相違がみられ, 病態解明の一助となることが示された.3. 胞状奇胎群において, きわめて盛んな細胞増殖状態が示され, またすべての例においてhypertetraploidyがみられた.4. 増殖期細胞の主要細胞はcytotrophoblastであり, HCG分泌機構との関連も示唆された.