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Single stranded DNA fragments in retinoblastoma patient blood plasma: link to oncogenesis and diagnostic validity

International Conference on Biotechnology, Biomarkers & Systems Biology
March 04-05, 2019 | Amsterdam, Netherlands

Kirill V. Ermakov and Alexander A. Bukhvostov

Pirogov Russian National Research Medical University, Russia

Posters & Accepted Abstracts: Biochem Mol biol J

Abstract:

A significant population of ultrashort (50n – 150n) single-stranded DNA fragments were found in exosome-free blood plasma of retinoblastoma patient (6.84 ng x mL-1), but not in plasma of healthy donors. An original HPLC technique has been employed. 5.0 year old male retinoblastoma (2A) patient and four same age/sex healthy donors were taken for blood plasma cfDNA extraction. A consequent treatment of DNA extract with exonucleases  and III, S1 nuclease, and proteinase K was followed then by a cascade ultrafiltration on K75/K25 SPM TechSep membranes (Mirabel, France). /III-nuclease resistant 25K – 75K compounds were analysed by size exclusion/anion exchange HPLC. For this purpose, its key parameters were estimated as the followings: stationary phase – polymethylamidopropylmethacrylamide; column PRP-X600 AE, 4.6 x 150.0mm, 5.0 particles, 1.6 meq/mL (Hamilton Corp., USA); 1,800 p.s.i., 22° – 25°C, 0.8 mL/min elution rate. Both synchronous linear elution LiCl2 (0 – 2.5M) and pH (8.0 – 4.0) gradients were formed on 100mM Tris/ acetonitrile (85:15, v/v). Waters/Hamilton compatible Breeze 200SLE Analytical System, W2998 UV-Detector (254nm), W600E gradient former (Waters, Inc., USA). Sample loading: 80 – 100g DNA in 50L 100mM Tris-HCl (pH 8.0)/ acetonitrile (85:15. v/v). As mentioned above, ssDNA short fragments were found in plasma of retinoblastoma patient. To the contrast, in control donors, a smaller population of ssDNA (2.40 – 2.82 ng x mL-1) was found consisting of essentially larger, 350n – 400n, sequences. A separation efficiency shown by our HPLC technique allows to reveal the size/charge – different populations within an ssDNA pool in cancer plasma which is not always possible in both PCR-based DNA size estimations and a routine agarose gel electrophoretic procedures. The later would mean a possible release of ssDNA directly in the “cancer-booming” DNA defects replacement. HPLC proposed is a simple and reliable tool for further epigenic and diagnostic studies on patients with retinoblastoma.

Biography :

Kirill V. Ermakov - Postgraduate Student, Department of Medical Nanobiotechnology Graduated from Pirogov Russian National Research Medical University of the Ministry of Health of the Russian Federation in 2017. Research interests: Spin-selective biochemistry, chemical enzymology, experimental oncology and pharmacology. Alexander A. Bukhvostov - Ph.D., Assistant professor of the Department of Medical Nanobiotechnologies. Graduated from I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University) in 2011. Research interests: Spin-selective biochemistry, chemical enzymology, experimental oncology and pharmacology.

E-mail: ermakovkv07@gmail.com tanzbukh@gmail.com