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


METHODS
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.


CONCLUSIONS
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 degrees C.
Holgado
Elaine
E
TDL Genetics, The Doctors Laboratory, London, UK. elaine.holgado@tdlpathology.com
Holgado
Ben
B
Liddle
Stuart
S
Ballard
Terry
T
Levett
Lisa
L
D002824:Chorionic Villi / Q000737:chemistry*; D015193:Chorionic Villi Sampling / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007621:Karyotyping / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D014314:Trisomy / Q000175:diagnosis / Q000235:genetics
D004247:DNA