The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content.: Implication for DNA sequencing by hybridization
The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content.: Implication for DNA sequencing by hybridization
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DOI：
10.1093/nar/26.18.4249
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发表时间：
1998-09-15
影响因子：
14.9
通讯作者：
Thuong, NT
中科院分区：
文献类型：
--
作者：
Nguyen, HK;Bonfils, E;Thuong, NT
通过最近报道的杂交技术进行测序需要形成具有相似稳定性的DNA双链体。在本文中，我们描述了一种新的策略，以获得DNA双链体的热稳定性独立于其AT/GC比含量。在给定长度和不同碱基组成的35个天然双链体和27个修饰双链体上获得熔解数据。与相应的天然化合物相比，用AT和/或G（4 Et）C碱基对构建的双链体表现出限制在较低温度范围内的热稳定性（16而不是51摄氏度），在用AT和/或G（4 Et）C碱基对构建的最不稳定和最稳定的双链体之间观察到的16 ℃的热稳定性差异主要是由于序列效应而不是它们的AT/G（因此，N-4-乙基-2 ′-脱氧胞苷（d（4 Et）C）与天然脱氧鸟苷特异性杂交，产生稳定性非常接近天然AT碱基对的G（4 Et）C碱基对。涉及d（4 Et）C的寡核苷酸探针可以通过使用亚磷酰胺化学的化学合成容易地制备。在DNA聚合酶存在下，用d（4 Et）CTP、dATP、dGTP和dTTP，通过随机引物或PCR技术成功地扩增了修饰的DNA靶。
Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G(4Et)C base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C), The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G(4Et)C base pairs is mainly due to the sequence effect and not to their AT/G(4Et)C ratio content, Thus N-4-ethyl-2'-deoxycytidine (d(4Et)C) hybridizes specifically with natural deoxyguanosine leading to a G(4Et)C base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d(4Et)C can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d(4Et)CTP, dATP, dGTP and dTTP in the presence of DNA polymerase, This new system might be very useful for DNA sequencing by hybridization.