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

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...

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Autores principales: Lahoud, Georges, Timoshchuk, Victor, Lebedev, Alexandre, Arar, Khalil, Hou, Ya-Ming, Gamper, Howard
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602760/
https://www.ncbi.nlm.nih.gov/pubmed/18987000
http://dx.doi.org/10.1093/nar/gkn797
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author Lahoud, Georges
Timoshchuk, Victor
Lebedev, Alexandre
Arar, Khalil
Hou, Ya-Ming
Gamper, Howard
author_facet Lahoud, Georges
Timoshchuk, Victor
Lebedev, Alexandre
Arar, Khalil
Hou, Ya-Ming
Gamper, Howard
author_sort Lahoud, Georges
collection PubMed
description 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.
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spelling pubmed-26027602009-03-05 Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine Lahoud, Georges Timoshchuk, Victor Lebedev, Alexandre Arar, Khalil Hou, Ya-Ming Gamper, Howard Nucleic Acids Res Chemistry and Synthetic Biology 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. Oxford University Press 2008-12 2008-11-05 /pmc/articles/PMC2602760/ /pubmed/18987000 http://dx.doi.org/10.1093/nar/gkn797 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Chemistry and Synthetic Biology
Lahoud, Georges
Timoshchuk, Victor
Lebedev, Alexandre
Arar, Khalil
Hou, Ya-Ming
Gamper, Howard
Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
title Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
title_full Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
title_fullStr Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
title_full_unstemmed Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
title_short Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine
title_sort properties of pseudo-complementary dna substituted with weakly pairing analogs of guanine or cytosine
topic Chemistry and Synthetic Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602760/
https://www.ncbi.nlm.nih.gov/pubmed/18987000
http://dx.doi.org/10.1093/nar/gkn797
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