Ehn Maria, Ahmadian Afshin, Nilsson Peter, Lundeberg Joakim, Hober Sophia
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Electrophoresis. 2002 Sep;23(19):3289-99. doi: 10.1002/1522-2683(200210)23:19<3289::AID-ELPS3289>3.0.CO;2-#.
Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.
焦磷酸测序是一种基于合成原理的DNA测序的四酶生物发光DNA测序技术。目前，该技术仅限于分析以单核苷酸多态性分析为代表的短DNA序列。为了扩大焦磷酸测序的应用范围，需要提高读取长度，并且已经努力纯化反应成分以及向焦磷酸测序反应中添加单链DNA结合蛋白（SSB）。在本研究中，我们通过比较103个独立互补DNA（cDNA）克隆的焦磷酸测序结果，系统地分析了SSB的作用。通过对焦磷酸测序图的深入分析，获得了关于具有不同特征的模板上低质量序列原因的更详细信息。此外，对单个cDNA克隆进行了实时生物传感器分析，以研究引物退火和SSB在这些模板上的结合情况。这些研究结果表明，在没有SSB的情况下焦磷酸测序性能良好的模板具有高效的引物退火和低SSB亲和力。还评估了通过提高引物退火效率来改善焦磷酸测序性能的替代策略。
