Comparing the Efficiency of Three Protocols in Isolation of Cell Free Fetal DNA From Maternal Blood

  • Fatemeh Karami Departement of Medical Genetics, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
  • Mohammad Reza Noori-Daloii Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Seddigheh Hantooshzadeh Vali-e-Asr Reproductive Health Research Center, School of Medicine, Tehran University of Medical Sciences, Iran
  • Mohammad Hossein Modarressi Departement of Medical Genetics, Science and Research Branch, Islamic Azad University, Tehran, Iran; Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Keywords: DNA, Isolation, NIPD, Maternal Blood, Protocol

Abstract

Objective: 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.

Author Biographies

Fatemeh Karami, Departement of Medical Genetics, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
Medical Genetics, Assistant Professor
Mohammad Reza Noori-Daloii, Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Medical Genetics, Professor
Seddigheh Hantooshzadeh, Vali-e-Asr Reproductive Health Research Center, School of Medicine, Tehran University of Medical Sciences, Iran
Reproductive Health Research Center, Professor
Mohammad Hossein Modarressi, Departement of Medical Genetics, Science and Research Branch, Islamic Azad University, Tehran, Iran; Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Medical Genetics, Professor

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Published
2018-03-06
How to Cite
1.
Karami F, Noori-Daloii MR, Hantooshzadeh S, Modarressi MH. Comparing the Efficiency of Three Protocols in Isolation of Cell Free Fetal DNA From Maternal Blood. JFRH. 11(3):146-51.
Section
Original Articles