biotechnology-biomarkers-systems-biology-2019-posters

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Page 44
Biochemistry & Molecular Biology Journal
ISSN: 2471-8084
Internat i ona l Conference on
Biotechnology, Biomarkers
& Systems Biology
M a r c h 0 4 - 0 5 , 2 0 1 9
Am s t e r d a m , N e t h e r l a n d s
Biotechnology, Biomarkers & Systems Biology 2019
Kirill V. Ermakov et al., Biochem Mol biol J 2019, Volume:5
DOI: 10.21767/2471-8084-C1-024
Asignificant 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.
Single stranded DNA fragments in retinoblastoma patient
blood plasma: link to oncogenesis and diagnostic validity
Kirill V. Ermakov and Alexander A. Bukhvostov
1Pirogov Russian National Research Medical University, Russia