Facile SNP detection using bifunctional, cross-linking oligonucleotide probes
Facile SNP detection using bifunctional, cross-linking oligonucleotide probes
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DOI：
10.1093/nar/gkn052
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发表时间：
2008-03-01
影响因子：
14.9
通讯作者：
Greenberg, Marc M.
中科院分区：
文献类型：
--
作者：
Peng, Xiaohua;Greenberg, Marc M.
开发了一种简便、灵敏的检测寡核苷酸特定序列的方法。检测DNA序列与单核苷酸的区分是通过结合选择性杂交与有效的交联反应。容易合成的双功能寡核苷酸探针含有修饰的嘧啶，能够在内部轻度氧化条件下形成链间交联，并在其5端生物素，用于区分质粒DNA中16-nt长位点的单个核苷酸差异。目的序列通过荧光光谱检测，利用亲和素和辣根过氧化物酶的偶联物在微量滴度板测定。该方法可以在不使用PCR的情况下检测到少至250 fmol的目标，并且显示出接近200:1的单核苷酸鉴别。原则上，这种方法能够探测任何含有2-脱氧腺苷的靶序列。
A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2-deoxyadenosine.