IMPROVING SEQUENCING ACCURACY WITH THERMOSTABLE PROTEINS

描述(改编自《调查者摘要》)：耐热性 酶是降低测序成本的一种很有前途的方法 基因组。鸟枪式测序策略，以及极大改进的方法 基于自动荧光DNA测序仪的测序，都有 帮助提高了全基因组测序的吞吐量。用于自动化 荧光DNA测序，对双链DNA进行测序的能力 可靠地通过以下方式提高DNA测序过程的效率 省去了亚克隆到M13载体的步骤。一个大问题 与双链DNA测序相关的似乎与 保持两个快速再退火互补的解离 同时允许对测序引物进行退火和延伸。 变性DNA链的重新结合导致较少的可读碱基。 初步实验表明，共价闭合的序列 在存在环状DNA的情况下，测序更稳健和准确 耐热DNA松弛拓扑异构酶。该效果是基于 这些酶对双螺旋结构的拓扑失稳(解离) 在广泛的离子条件下(Slesarev等人自然364：735-7， 1993年)。耐热拓扑异构酶解开超螺旋DNA 通过过早地减少DNA的数量来改进DNA作为测序模板 与真正的双脱氧末端竞争的终止分子。这 减少了背景频带，提高了基地呼叫的准确性。在……里面 此外，模板中的发夹结构被认为是 如释重负，消除了“强力止步”神器。进一步，改进 变性似乎提高了底物退火的效率， 增加测序信号。结论将在 DNA测序新方法与传统方法的比较形式 关于测序、信号强度和DNA消耗的保真度。

DESCRIPTION (Adapted from the Investigator's Abstract): Thermostable enzymes represent a promising approach to decreasing the cost of sequencing genomes. Shotgun sequencing strategies, and greatly improved methods of sequencing based on the automated fluorescent DNA sequencers, all have helped increase throughput for whole genome sequencing. For automated fluorescent DNA sequencing, the ability to sequence double stranded DNA reliably would make the process of DNA sequencing more efficient by eliminating the steps of subcloning into M13 vectors. A major problem associated with double-stranded DNA sequencing appears to be related to maintaining the dissociation of the two rapidly reannealing complementary strands while allowing annealing and extension of the sequencing primer. The reassociation of denatured DNA strands results in fewer readable bases. The preliminary experiments indicate sequencing of covalently closed circular DNA is more robust and accurate when sequenced in the presence of thermostable DNA-relaxing topoisomerases. The effect is based on the topological destabilization (unwinding) of the double helix by these enzymes in a wide range of ionic conditions (Slesarev et al. Nature 364:735-7, 1993). Unwinding supercoiled DNA by thermostable topoisomerases appears to improve DNA as a sequencing template by decreasing the number of prematurely terminated molecules which compete with true dideoxy terminations. This reduces background bands and increases the accuracy of base calling. In addition, it is thought that hairpin structures in the template are relieved, eliminating the "strong stop" artifact. Further, improved denaturation seems to increase the efficiency of primer annealing, increasing the sequencing signal. The conclusions will be presented in the form of comparison of traditional and new methods of DNA sequencing with respect to the fidelity of sequencing, signal intensity and DNA consumption.