Peng Xiaohua, Greenberg Marc M
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Nucleic Acids Res. 2008 Mar;36(5):e31. doi: 10.1093/nar/gkn052. Epub 2008 Feb 16.
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.
开发了一种简便、灵敏的检测寡核苷酸特定序列的方法。通过将杂交的选择性与高效的交联反应相结合，实现了具有单核苷酸区分能力的DNA序列检测。使用易于合成的双功能寡核苷酸探针，该探针含有一个修饰的嘧啶，能够在温和氧化条件下在内部形成链间交联，并且在其5'-末端带有生物素，用于区分质粒DNA中相差一个单核苷酸的16个核苷酸长的位点。在微量滴定板分析中，通过利用抗生物素蛋白和辣根过氧化物酶的缀合物，通过荧光光谱法检测目标序列。该方法无需使用PCR就能检测低至250飞摩尔的目标，并且具有接近200:1的单核苷酸区分能力。原则上，该方法能够探测任何含有2'-脱氧腺苷的目标序列。
