Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine.
Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine.
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DOI：
10.1093/nar/gkn797
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发表时间：
2008-12
影响因子：
14.9
通讯作者：
Gamper H
中科院分区：
文献类型：
--
作者：
Lahoud G;Timoshchuk V;Lebedev A;Arar K;Hou YM;Gamper H
一种直接的将常规DNA转化为伪互补DNA的酶促方法可以通过产生容易杂交的无结构靶标来提高寡核苷酸微阵列的性能。在这里，我们筛选了几个高度不稳定的G和C类似物，其中一个可以与2-氨基腺嘌呤(NA)和2-硫代胸腺嘧啶(ST)一起产生完全可以被互补探针访问的无结构DNA。这些类似物包括生物活性碱基如6-硫代鸟嘌呤(SG)、5-硝基胞嘧啶(NitroC)、2-嘧啶酮(P；ZeBularine的游离碱)和6-甲基呋喃并嘧啶酮(MefP)，它们被制备成dNTPs，并被评估为缺乏编辑功能的T7和Phi29 DNA聚合酶的底物。通过使用修饰的寡核苷酸或引物延伸产物的溶液杂交试验来表征类似物的配对特性。P和MEP不支持强扩增，而SG和NitroC支持。然而，在杂交分析中，SG缺乏辨别力，而NitroC与C配对太强。另外两个碱基类似物7-硝基-7-去氮次黄嘌呤(NitrocH)和2-硫胞嘧啶(SC)的dNTPs表现出最大的潜力。任何一种类似物都可以与NA和ST一起使用来产生几乎无结构的DNA。将探针杂交到这些修饰的DNA将需要开发与NitrocH或SC强烈配对的碱基类似物。
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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影响因子：
14.9
作者：
MARTIN, FH;CASTRO, MM;TINOCO, I
通讯作者：
TINOCO, I
影响因子：
14.9
作者：
Hacia, JG;Woski, SA;Collins, FS
通讯作者：
Collins, FS
影响因子：
14.9
作者：
MIZUSAWA, S;NISHIMURA, S;SEELA, F
通讯作者：
SEELA, F
影响因子：
15
作者：
Kushon, SA;Jordan, JP;Armitage, BA
通讯作者：
Armitage, BA
影响因子：
1.3
作者：
McDougall, MG;Sun, L;Kumar, S
通讯作者：
Kumar, S