Enhanced detection and distinction of RNA by enzymatic probe ligation
Enhanced detection and distinction of RNA by enzymatic probe ligation
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DOI：
10.1038/77367
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发表时间：
2000-07-01
影响因子：
46.9
通讯作者：
Landegren, U
中科院分区：
文献类型：
--
作者：
Nilsson, M;Barbany, G;Landegren, U
重要的是，代表基因家族成员的RNA分子在表达分析中被区分开，并且可能需要更大的分辨能力来鉴定转录本的等位基因变体，以便研究印记或研究突变基因在组织中的分布。连接酶介导的基因检测允许精确区分DNA序列变体1、2、3、4，但尚不清楚连接酶是否也可用于区分RNA序列的变体。在这里，我们提出的条件下，有效连接的DNA寡核苷酸对杂交旁边的RNA链，允许歧视的任何单核苷酸探针靶错配的20- 200倍之间的一个因素。该机制允许挂锁探针用于区分RNA中的单核苷酸变体。因此，连接酶介导的基因检测可以提供高灵敏度和准确的连接酶介导的检测和区分RNA序列变体在溶液中，在DNA微阵列上，和在原位。
It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants 1, 2, 3, 4, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe–target mismatch by a factor of between 20-and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.