A UNIVERSAL NUCLEOSIDE FOR USE AT AMBIGUOUS SITES IN DNA PRIMERS
A UNIVERSAL NUCLEOSIDE FOR USE AT AMBIGUOUS SITES IN DNA PRIMERS
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DOI：
10.1038/369492a0
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发表时间：
1994-06-09
期刊：
影响因子：
64.8
通讯作者：
BERGSTROM, DE
中科院分区：
文献类型：
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作者：
NICHOLS, R;ANDREWS, PC;BERGSTROM, DE
无差别碱基类似物或通用碱基将是寡核苷酸探针和引物的宝贵组分，用于解决由于遗传密码简并性或当仅可获得片段肽序列数据时出现的设计问题。我们设计了一种替代以前的通用核苷候选物（1-9）的新类似物，1-（2 '-脱氧-β-D-呋喃核糖基）-3-硝基吡咯（指定为M;图1），它使堆积最大化，同时使氢键相互作用最小化，而不会空间破坏DNA双链体。在几个位点含有M的寡核苷酸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 ′）与精确匹配d（5 ′-CGT AAT CAG AAA ACA AT-3 ′）一样有效。也可以使用在几个连续位置含有M的引物进行测序，例如d（5 ′-CGT AAT MMM MMM MMM AT-3 ′）。解链曲线表明，在序列d（5 ′; CCT TTT TMT TTT TGG-3 ′）和d（5 ′-CCA AAA AXA AAA AGG-3 ′）杂交时形成的双链体，其中X是A、C、G或T，其解链温度低于仅含d（A.T）和d（C.G）碱基对的相应双链体，但在不同X碱基之间几乎没有变化（T-m范围3 ℃）。
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(1-9), 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 (T-m range 3 degrees C).