Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine.

Abstract:

:A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Lahoud G,Timoshchuk V,Lebedev A,Arar K,Hou YM,Gamper H

doi

10.1093/nar/gkn797

subject

Has Abstract

pub_date

2008-12-01 00:00:00

pages

6999-7008

issue

22

eissn

0305-1048

issn

1362-4962

pii

gkn797

journal_volume

36

pub_type

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