Comparing the Efficiency of Three Protocols in Isolation of Cell Free Fetal DNA From Maternal Blood
AbstractObjective: Recent advances in non-invasive prenatal diagnosis (NIPD) through cell free fetal DNA (cffDNA) has highlighted cffDNA purification as a critical initial step. Herein, we aimed to compare the efficiency of one proposed protocol with two commercial kits for isolation of cffDNA.Materials and methods: cffDNA was isolated from whole blood of 50 normal pregnancies using one proposed manual protocol compared with QIAamp DNA Blood Mini and Bioneer Kits. Methylated DNA immunoprecipitation real time polymerase chain reaction (MeDIP-Real time PCR) was performed to quantify three fetal specific sequences.Results: Maximum cffDNA quantity was obtained by suggested protocol (248.79 ± 14.07 ng/µl) and the best quality was achieved by Bioneer Kit (OD ratio: 260/280 nm/nm: 1.69 ± 0.09, 260/230 nm/nm: 1.15 ± 0.13) (p < 0.001). Enrichment of fetal specific sequences was significantly higher when proposed protocol was used to isolate cffDNA (p = 0.01).Conclusion: Inhibitory effect of NaI on nucleases and double digestion of DNA associated proteins may be the main reasons behind the superiority of suggested protocol. Significantly higher amplification of fetal specific sequences in suggested protocol would be a strong evidence on recovery of small fetal fragments as demonstrated with its maximum total DNA quantity and amplification in different PCR reactions.
Grootkerk-Tax MG, Soussan AA, de Haas M, Maaskant-van Wijk PA, van der Schoot CE. Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma. Transfusion 2006; 46: 2142-8.
Akolekar R, Farkas DH, VanAgtmael AL, Bombard AT, Nicolaides KH. Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation. Prenat Diagn 2010; 30: 918-23.
Yu H, Shen Y, Ge Q, He Y, Qiao D, Ren M, et al. Quantification of maternal Serum cell-free fetal DNA in early-onset preeclampsia. Int J Mol Sci 2013; 14: 7571-82.
Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999; 64: 218-24.
Chan KC, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004; 50: 88-92.
Dhallan R, Au WC, Mattagajasingh S, Emche S, Bayliss P, Damewood M, et al. Methods to increase the percentage of free fetal DNA recovered from the maternal circulation. JAMA 2004; 291: 1114-9.
Dhallan R, Guo X, Emche S, Damewood M, Bayliss P, Cronin M, et al. A non-invasive test for prenatal diagnosis based on fetal DNA present in maternal blood: a preliminary study. Lancet 2007; 369: 474-81.
Jorgez CJ, Dang DD, Simpson JL, Lewis DE, Bischoff FZ. Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women. Genet Med 2006; 8: 615-9.
Modarressi MH, Cameron J, Taylor KE, Wolfe J. Identification and characterisation of a novel gene, TSGA10, expressed in testis. Gene 2001; 262: 249-55.
Tsaliki E, Papageorgiou EA, Spyrou C, Koumbaris G, Kypri E, Kyriakou S, et al. MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21. Prenat Diagn 2012; 32: 996-1001.
Skaanild MT, Clausen J. Method for estimation of benzo[a]pyrene DNA adducts. Anal Biochem 1992; 205: 337-41.
Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol 1995; 27: 647-53.
Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci U S A 1997; 94: 514-9.
Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990; 28: 495-503.
Repiska G, Sedlackova T, Szemes T, Celec P, Minarik G. Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma. Clin Chem Lab Med 2013; 51: 1185-9.
Clausen FB, Krog GR, Rieneck K, Dziegiel MH. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit. Prenat Diagn 2007; 27: 6-10.
Fleischhacker M, Schmidt B, Weickmann S, Fersching DM, Leszinski GS, Siegele B, et al. Methods for isolation of cell-free plasma DNA strongly affect DNA yield. Clin Chim Acta 2011; 412: 2085-8.
Kirsch C, Weickmann S, Schmidt B, Fleischhacker M. An improved method for the isolation of free-circulating plasma DNA and cell-free DNA from other body fluids. Ann N Y Acad Sci 2008; 1137: 135-9.
Keshavarz Z, Moezzi L, Ranjbaran R, Aboualizadeh F, Behzad-Behbahani A, Abdullahi M, et al. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum. Avicenna J Med Biotechnol 2015; 7: 85-8.
Breitbach S, Tug S, Helmig S, Zahn D, Kubiak T, Michal M, et al. Direct quantification of cell-free, circulating DNA from unpurified plasma. PLoS One 2014; 9: e87838.
Schmidt B, Weickmann S, Witt C, Fleischhacker M. Improved method for isolating cell-free DNA. Clin Chem 2005; 51: 1561-3.