Direct Assessment of Single-Cell DNA Using Crudely Purified Live Cells: A Proof of Concept for Noninvasive Prenatal Definitive Diagnosis

Publication date: February 2020Source: The Journal of Molecular Diagnostics, Volume 22, Issue 2Author(s): Taisuke Sato, Yuki Ito, Osamu Samura, Hiroaki Aoki, Toru Uchiyama, Aikou Okamoto, Kenichiro HataNoninvasive testing techniques are often used for fetal diagnosis of genetic abnormalities but are limited by certain characteristics, including noninformative results. Thus, novel methods of noninvasive definitive diagnosis of fetal genetic abnormalities are needed. The aim of this study was to develop a single-cell DNA analysis method with high sensitivity and specificity that enables direct extraction of genetic information from live fetal cells in a crude mixture for simultaneous evaluation. Genomic DNA from circulating fetal CD45−CD14− cells, an extremely rare cell type, extracted from 10-mL samples of maternal peripheral blood, was extracted using a single-cell–based droplet digital (sc-dd) PCR system with a modified amount of polymerase. A hexachloro-6-carboxyfluorescein–labeled RPP30 probe was used as an internal control and a 6-carboxyfluorescein–labeled SRY probe as a target. The results indicated that no droplets generated with samples from pregnant women carrying female fetuses were positive for both probe signals, whereas droplets prepared with samples from pregnant women carrying male fetuses were positive for both probe signals. The latter was considered a direct assessment of genetic information from single circulating male fetal cells. Thus, the modif...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research