Ronaghi M
Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, California 94304, USA.
Anal Biochem. 2000 Nov 15;286(2):282-8. doi: 10.1006/abio.2000.4808.
In modern biology, there is a critical need to develop a high-throughput and inexpensive platform for DNA sequencing. Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. In these studies, single-stranded DNA-binding protein (SSB) was added to the primed DNA template prior to the Pyrosequencing reaction. The addition of SSB to a Pyrosequencing reaction system resulted in a read length of more than 30 nucleotides. Improvements were observed as: (i) increased efficiency of the enzymes, (ii) reduced mispriming, as measured by nonspecific signals, (iii) an increase in signal intensity during the reaction, (iv) higher accuracy in reading the number of identical adjacent nucleotides in difficult templates, and (v) longer reads. The usefulness of these results for future Pyrosequencing applications is discussed.
在现代生物学中，迫切需要开发一种用于DNA测序的高通量且低成本的平台。焦磷酸测序是一种非电泳单管DNA测序方法，它利用四种酶之间的协同作用来监测DNA合成。在这些研究中，在焦磷酸测序反应之前，将单链DNA结合蛋白（SSB）添加到引物化的DNA模板中。向焦磷酸测序反应体系中添加SSB导致读长超过30个核苷酸。观察到的改进包括：（i）酶效率提高；（ii）以非特异性信号衡量的错配减少；（iii）反应过程中信号强度增加；（iv）在困难模板中读取相同相邻核苷酸数量时准确性更高；（v）读长更长。讨论了这些结果对未来焦磷酸测序应用的有用性。
