Wang Jinke, Bai Yunfei, Li Tongxiang, Lu Zuhong
National Laboratory for Molecular and Biomolecular Electronics, Southeast University, Nanjing 210096 Jiangsu Province, China.
J Biochem Biophys Methods. 2003 Mar 28;55(3):215-32. doi: 10.1016/s0165-022x(03)00048-4.
We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.
我们制备了双链DNA（dsDNA）微阵列，其中单分子发夹双链DNA探针固定在载玻片上。单分子发夹双链DNA微阵列的制备分四个步骤：首先，合成在3'羟基末端具有两个反向互补序列且在5'氨基末端具有突出序列的单链DNA（ssDNA）寡核苷酸。其次，将ssDNA微点样到戊二醛衍生的载玻片上以形成ssDNA微阵列。第三，使两个反向互补序列退火，在固定化ssDNA的3'末端形成发夹引物，从而创建部分双链DNA微阵列。第四，通过酶促延伸发夹引物，将部分双链DNA微阵列转化为完整的双链DNA微阵列。通过在Klenow延伸中掺入荧光标记的dUTP以及用限制性内切酶消化双链DNA微阵列，证明了酶促合成的优异效率和高精度。通过将Cy3标记的NF-κB与双链DNA微阵列结合，验证了与双链DNA微阵列结合的DNA结合蛋白的可及性和特异性。双链DNA微阵列具有巨大潜力，可为研究基因表达调控、限制等过程中涉及的序列特异性DNA/蛋白质相互作用提供高通量平台。
