Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women.
OBJECTIVE
Methods to isolate cell-free fetal DNA from maternal plasma are critical in developing noninvasive fetal DNA testing strategies. Given that plasma consists of heterogeneous DNA-size fragments in a complex mix of proteins, recovery and analysis of this DNA are understandably inefficient. To facilitate recovery, we performed qualitative and quantitative analysis of DNA isolated from maternal plasma.


METHODS
DNA isolated from maternal blood (n = 15) was compared using five different DNA isolation protocols: two conventional, two column-based, and one magnetic-bead based. Purity and concentration of DNA recovered were determined with a NanoDrop spectrophotometer. Real-time polymerase chain reaction quantification of the beta-globin and DYS1 loci was performed to determine total and fetal-specific genome equivalents, respectively.


RESULTS
DNA quality and quantity were different among the five methods tested. Although purity and concentration of total DNA were greatest with the conventional boiling-lysis approach, correct detection of a male fetus was achieved in only 62.5% of cases. DNA isolation using the magnetic beads yielded the highest quantity of total DNA (2018.83 +/- 4.09 GEq/mL), with 100% fetal DNA detection.


CONCLUSIONS
Optimal plasma DNA recovery protocols must take into account DNA purity and concentration. We confirm that the magnetic-beads method provides a fast, simple, sensitive, and specific approach to purify plasma DNA. The resulting high-quality DNA facilitates efficient examination of fetal DNA sequences.
Jorgez
Carolina J
CJ
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77054, USA.
Dang
Dianne D
DD
Simpson
Joe Leigh
JL
Lewis
Dorothy E
DE
Bischoff
Farideh Z
FZ
D004247:DNA / Q000302:isolation & purification*; D005260:Female; D052203:Ferrosoferric Oxide / Q000737:chemistry; D005821:Genetic Techniques; D006801:Humans; D008863:Microspheres; D011247:Pregnancy / Q000097:blood*
D004247:DNA; D052203:Ferrosoferric Oxide