基于核酸保护原理的 DNA芯片检测技术

The Nucleic Acid Analysis by DNA Chip Technique Based on Nuclease S1 Protection

制备出 3′-末端与载玻片交联、 5′-末端用 32P标记的检测 DNA的芯片，方法以化学法合成了3′-末端为尿嘧啶核糖的寡聚脱氧核糖核苷酸片段。用 32P标记寡核苷酸的 5′-末端，经过高碘酸氧化后与玻璃基片表面的脂肪胺基缩合，并用硼氢化钠还原，制成寡核苷酸 3′-末端与玻片共价交联、 5′-末端为同位素标记的 DNA芯片。将该芯片与液相中的核酸片段杂交，再用核酸酶 S1酶切。结果当液相中的核酸与玻片上共价交联的寡核苷酸片段产生特异性杂交配对时，核酸酶 S1不能酶切玻片上的寡核苷酸片段，且玻片上被保护的寡核苷酸量与液相中的与它配对的核酸量之间呈线性相关（ r=0.9967,P< 0.001, n=7)。结论上述检测法可以定性和定量地检测液相中的核酸。由于该法制备的 DNA芯片各个位点的 DNA探针的含量为已知，待测的核酸样品无需进行同位素或荧光标记，操作简便，适用于对样品的 DNA或 RNA进行检测。

Objective Establishing a method for quantitative analysis of nucleic acid by DNA chips. Methods The modified oligonucleotides with ribose at 3′- end was chemically synthesized. The 5′- end was labeled by radioisotope 32P with kinase catalyzed reactions. Such oligonucleotides were converted into di- aldehyde at 3′- end by oxidization with NaIO4, and then were spotted on glass slide with the amino group modified surface. After reduced with NaBH4, the oligonucleotides were attached strongly. The DNA chips prepared with this method were hybridized with nucleic acids existed in the solution and then digested with nuclease S1. Results When they were paired with the nucleic acids in the solution perfectly, the oligonucleotides on the chip were not cleaved by nuclease S1. Otherwise, the oligonucleotides on chip were cleaved. The protection efficiencies appeared proportional to the perfect paired nucleic acids in the solution when the content of target nucleic acids were less than the spots on the slides. Conclusions The method was developed for both qualitative and quantitative analysis of nucleic acid. As it was not required to label the samples with radioisotope or fluorescence, it might be a practical choice for clinical tests.