A novel method for extracting DNA from chorionic villus samples for use in CVS-PCR, which ensures complete villus dissociation

• Published in: Prenatal diagnosis Vol. 29; no. 2; pp. 113 - 119
• Main Authors: Holgado, Elaine, Holgado, Ben, Liddle, Stuart, Ballard, Terry, Levett, Lisa
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.02.2009; Wiley
• Subjects: Biological and medical sciences; Chorionic Villi - chemistry; Chorionic Villi Sampling - methods; CVS; Delivery. Postpartum. Lactation; DNA - isolation & purification; DNA extraction; Female; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; Gynecology. Andrology. Obstetrics; Humans; Karyotyping - methods; Medical sciences; Molecular and cellular biology; mosaicism; placental mosaicism; Polymerase Chain Reaction - methods; Pregnancy; prenatal diagnosis; quantitative fluorescent PCR; Trisomy - diagnosis; Trisomy - genetics; CVS; DNA extraction; Sample; Gynecology; Neonatology; Method; Obstetrics; Polymerase chain reaction; placental mosaicism; Prenatal diagnosis; Placenta; DNA; Genetics; Mosaicism; Extraction; Molecular biology; Chorionic villi; quantitative fluorescent PCR; Quantitative analysis
• ISSN: 0197-3851; 1097-0223
• DOI: 10.1002/pd.2160

Objective To demonstrate that glass disruption beads dissociate chorionic villus samples releasing DNA from mesenchymal and cytotrophoblast cells that is suitable for processing by CVS–PCR (rapid molecular aneuploidy testing). This method is quicker than conventional methods and may limit discrepancies between PCR and karyotype in certain types of placental mosaicism. Method DNA was extracted from villus samples by mechanical disruption of the cells using glass beads. This method was compared to collagenase incubation followed by chelex extraction of the digested villus. PCR data generated were compared using standard criteria. Results DNA extracted by glass bead disruption generated data of equivalent quality to that obtained from DNA extracted using conventional collagenase and chelex‐based extraction method. The case study demonstrates probable cytotrophoblast enrichment of a sample when processed by collagenase digestion and chelex incubation. Re‐extraction of the digested sample by glass bead disruption resulted in cytotrophoblast and mesenchyme cells contributing to the supernatant. Conclusion Glass bead disruption of chorionic villus samples is an effective, inexpensive and rapid DNA extraction method that dissociates villus ensuring that DNA from both cytotrophoblast and mesenchyme cells is represented in the supernatant. Extracted DNA produced is suitable for CVS–PCR and can be stored stably at − 20 °C. Copyright © 2008 John Wiley & Sons, Ltd.