Nichols R, Andrews P C, Zhang P, Bergstrom D E
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Nature. 1994 Jun 9;369(6480):492-3. doi: 10.1038/369492a0.
A non-discriminatory base analogue, or universal base, would be an invaluable component of oligonucleotide probes and primers for solving the design problems that arise as a result of the degeneracy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates, a new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides containing M at several sites were used as primers for sequencing and the polymerase chain reaction. The sequencing primer d(5'-CGT AAM CAM AAM ACM AT-3') is as effective as the exact match d(5'-CGT AAT CAG AAA ACA AT-3'). It is also possible to sequence using a primer containing M at several contiguous positions, for example d(5'-CGT AAT MMM MMM MMM AT-3'). Melting curves show that duplexes formed on hybridization of the sequences d(5'-CCT TTT TMT TTT TGG-3') and d(5'-CCA AAA AXA AAA AGG-3'), where X is A, C, G or T, melted at a lower temperature than the corresponding duplexes containing only d(A.T) and d(C.G) base pairs, but showing little variation among different X bases (Tm range 3 degrees C).
一种非歧视性碱基类似物，即通用碱基，将是寡核苷酸探针和引物的一个宝贵组成部分，可用于解决因遗传密码简并性或仅获得片段化肽序列数据时出现的设计问题。我们设计了一种替代先前通用核苷候选物的新类似物，即1-(2'-脱氧-β-D-呋喃核糖基)-3-硝基吡咯（命名为M；图1），它能在不破坏DNA双链空间结构的情况下，使堆积最大化，同时使氢键相互作用最小化。含有多个位点M的寡核苷酸被用作测序和聚合酶链反应的引物。测序引物d(5'-CGT AAM CAM AAM ACM AT-3')与完全匹配的引物d(5'-CGT AAT CAG AAA ACA AT-3')效果相同。使用在几个连续位置含有M的引物进行测序也是可行的，例如d(5'-CGT AAT MMM MMM MMM AT-3')。熔解曲线表明，序列d(5'-CCT TTT TMT TTT TGG-3')和d(5'-CCA AAA AXA AAA AGG-3')（其中X为A、C、G或T）杂交形成的双链，其熔解温度低于仅含d(A.T)和d(C.G)碱基对的相应双链，但不同X碱基之间变化不大（熔解温度范围为3℃）。
