Genome Institute of Singapore, 60 Biopolis Street, 02-01 Genome, Singapore 138672, Singapore.
Biochem Biophys Res Commun. 2010 Oct 8;401(1):117-23. doi: 10.1016/j.bbrc.2010.09.022. Epub 2010 Sep 15.
Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid and real-time sequence determination. Although ample genomic research has been undertaken using pyrosequencing, the requirement of relatively high amount of DNA template and the difficulty in sequencing the homopolymeric regions limit its key advantages in the applications directing towards clinical research. In this study, we demonstrate that pyrosequencing on homopolymeric regions with 10 identical nucleotides can be successfully performed with optimal amount of DNA (0.3125-5 pmol) immobilized on conventional non-porous Sepharose beads. We also validate that by using porous silica beads, the sequencing signal increased 3.5-folds as compared to that produced from same amount of DNA immobilized on solid Sepharose beads. Our results strongly indicate that with optimized quantity of DNA and suitable solid support, the performance of pyrosequencing on homopolymeric regions and its detection limit has been significantly improved.
焦磷酸测序是一种基于合成测序的 DNA 测序技术，能够实现快速实时的序列测定。尽管已经有大量的基因组研究使用焦磷酸测序，但相对较高量的 DNA 模板的要求以及测序同聚物区域的困难限制了其在临床研究应用中的关键优势。在本研究中，我们证明了在含有 10 个相同核苷酸的同聚物区域上，使用优化量的（0.3125-5 pmol）固定在常规非多孔 Sepharose 珠上的 DNA 可以成功地进行焦磷酸测序。我们还验证了，与相同量的 DNA 固定在固体 Sepharose 珠上相比，使用多孔硅胶珠可以将测序信号提高 3.5 倍。我们的结果强烈表明，通过优化 DNA 的量和合适的固相支持，焦磷酸测序在同聚物区域的性能及其检测限得到了显著提高。
