Summary
Circulating cell-free fetal deoxyribonucleic acids (cffDNAs) are promising biomarkers with various potential clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNAs. Further improvements to the original protocol for the extraction of cffDNAs from AF supernatant resulted in statistically significant higher yields of high-quality cffDNAs, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g., comparative genomic hybridization microarrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples compared with the original protocol, the current method, using large silico-membranes, allows for the extraction of cffDNAs from up to 10 samples in <3 h. The replacement of the original lysis buffer eliminates the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol (e.g., to reduce potential contamination). The improvements in the yield with the current protocol make it possible to augment current standard of care through the analysis of this previously unappreciated source of genetic material. Furthermore, the improvements allow for exploration of widely unknown genetic, pathophysiological, and kinetic issues of cell-free fetal DNA in AF.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Lo, Y. M. D, Corbetta, N., Chamberlain, P. F., et al. (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485–487.
Zhong, X. Y., Laivuori, H., Livingston, J. C., et al. (2001) Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am. J. Obstet. Gynecol. 184, 414–419.
Wataganara, T., LeShane, E.S., Farina, A., et al. (2003) Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13, but not trisomy 18. Hum. Genet. 112, 204–208.
Farina, A., LeShane, E. S., Lambert-Messerlian, G. M., Canick, J. A., Lee, T., and Neveux, L. M. (2003) Valuation of fetal cell free DNA as a second-trimester marker of Down syndrome pregnancy. Clin. Chem. 49, 239–242.
Bianchi, D. W., LeShane, E., and Cowan, J. M. (2001) Large amounts of cell-free fetal DNA are present in amniotic fluid. Clin. Chem. 47, 1867–1869.
Larrabee, P. B., Johnson, K. L., Pestova, E., et al. (2004) Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype. Am. J. Hum. Genet. 75, 485–491.
Lapaire, O., Stroh, H., Peter, I., et al. (2006) Larger filters and change of lysis buffer significantly improve the quantity of extracted cell-free DNA from amniotic fluid. Clin. Chem. 52, 156–157.
Acknowledgement
We thank Helene Stroh, Inga Peter, and Janet Cowan with the team of the Tufts-New England Medical Center, Boston, MA, for advice. This work was supported by National Institutes of Health grant R01HD42053 and Swiss National Fund PBBSB-108590.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lapaire, O., Johnson, K.L., Bianchi, D.W. (2008). Method for Extraction of High-Quantity and -Quality Cell-Free DNA from Amniotic Fluid. In: Hahn, S., Jackson, L.G. (eds) Prenatal Diagnosis. Methods in Molecular Biology™, vol 444. Humana Press. https://doi.org/10.1007/978-1-59745-066-9_24
Download citation
DOI: https://doi.org/10.1007/978-1-59745-066-9_24
Publisher Name: Humana Press
Print ISBN: 978-1-58829-803-4
Online ISBN: 978-1-59745-066-9
eBook Packages: Springer Protocols