重组大肠杆菌单链结合蛋白性质表征及其在提高焦磷酸测序准确性中的应用

Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing

摘要:

利用基因工程手段表达了分子量约为24 kDa的重组大肠杆菌单链结合蛋白 (r-SSBP)，通过凝胶阻滞电泳与DNA熔解温度 (Tm) 影响实验表征了r-SSBP与单链DNA (ssDNA) 结合的特性，结果表明，r-SSBP可以与ssDNA结合，并且能够降低DNA的Tm值，同时还能增大含有单个错配碱基的DNA与完全匹配的DNA的Tm值差异，这一特性在提高单核苷酸多态性检测的特异性方面具有潜在的应用价值。此外，将r-SSBP应用于本课题组开发的高灵敏度焦磷酸测序体系中测定已知序列ssDNA模板，结果表明，r

Abstract:

We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (Tm) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the Tm of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the Tm difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.