Xu Y, Kool E T
Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
Nucleic Acids Res. 1999 Feb 1;27(3):875-81. doi: 10.1093/nar/27.3.875.
The success of oligonucleotide ligation assays in probing specific sequences of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe here a study of the effect of mismatches on a new non-enzymatic, reagent-free method for ligation of oligonucleotides. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5'-end iodide with a 3'-phosphorothioate group. The data show that this ligation proceeds somewhat more slowly than ligation by T4 ligase, but with substantial discrimination against single base mismatches both at either side of the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are observed in the latter case. Experiments at varied concentrations and temperatures are carried out both with the autoligation of two adjacent linear oligonucleotides and with intramolecular autoligation to yield circular 'padlock' DNAs. Application of optimized conditions to discrim-ination of an H- ras codon 12 point mutation is demonstrated with a single-stranded short DNA target.
寡核苷酸连接测定法在探测特定DNA序列方面取得成功，很大程度上源于对连接位点碱基错配具有高度的酶选择性。我们在此描述了一项关于错配如何影响一种新的非酶促、无需试剂的寡核苷酸连接方法的研究。在这种方法中，两条结合在互补链相邻位点的寡核苷酸通过用3'-硫代磷酸基团取代5'-末端碘化物而进行自动连接。数据表明，这种连接比T4连接酶介导的连接进行得稍慢一些，但对连接位点两侧以及其中一个寡核苷酸结合位点内几个核苷酸处的单碱基错配具有显著的区分能力。在后一种情况下，观察到对单个错配的选择性大于100倍。我们对两条相邻线性寡核苷酸的自动连接以及分子内自动连接以产生环状“锁式”DNA都进行了不同浓度和温度下的实验。通过单链短DNA靶标证明了将优化条件应用于区分H- ras密码子12点突变的情况。
