2017  0,977
2016  0,799
2015  0,662
2014  0,740
2013  0,739
2012  0,637
2011  0,658
2010  0,654
2009  0,570
2008  0,849
2007  0,805
2006  0,330
2005  0,435
2004  0,623
2003  0,567
2002  0,641
2001  0,490
2000  0,477
1999  0,762
1998  0,785
1997  0,507
1996  0,518
1995  0,502
Vol 52(2018) N 6 p. 854-864; DOI 10.1134/S0026893318060110 Full Text

S.A. Lapa1*, K.S. Romashova1, M.A. Spitsyn1,2, V.E. Shershov1, V.E. Kuznetsova1, T.O. Guseinov1,2, O.A. Zasedateleva1, S.P. Radko2,3, E.N. Timofeev1, A.V. Lisitsa3, A.V. Chudinov1,2

Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation

1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia
2IBMC-EcoBioPharm Ltd., Moscow, 119121 Russia
3Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences, Moscow, 119121 Russia

Received - 2017-11-26; Accepted - 2017-12-05

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

combinatorial DNA libraries, modified nucleotides, 2'-deoxyuridine-5'-triphosphate, PCR in inverse emulsion, DNA polymerases, modified aptamers