IMPROVING SEQUENCING ACCURACY WITH THERMOSTABLE PROTEINS

描述（改编自研究者摘要）：耐热 酶代表了降低测序成本的有希望的方法 基因组 鸟枪测序策略，以及大大改进的方法， 基于自动荧光DNA测序仪的测序， 有助于提高全基因组测序的吞吐量。 用于自动 荧光DNA测序，测序双链DNA的能力 可靠地将使DNA测序过程更有效， 省去了亚克隆入M13载体的步骤。 一个主要问题 与双链DNA测序相关的，似乎与 维持两个快速再退火互补的 链，同时允许测序引物退火和延伸。 变性DNA链的重新结合导致更少的可读碱基。 初步实验表明，共价闭合的 当在存在下测序时，环状DNA更稳健和准确。 热稳定DNA松弛拓扑异构酶。 效果是基于 通过这些酶使双螺旋的拓扑不稳定（解旋 在宽范围的离子条件下（Slesarev等，Nature 364：735-7， 1993年）。 通过热稳定的拓扑异构酶解超螺旋DNA， 通过减少过早的DNA序列的数目来改进DNA作为测序模板， 与真正的双脱氧末端竞争的封端分子。 这 减少了背景条带并提高了碱基识别的准确性。 在 此外，认为模板中的发夹结构是 松了一口气，消除了“强停”神器。 此外，改善 变性似乎增加了引物退火的效率， 增加测序信号。 结论将载于 比较传统和新的DNA测序方法， 在测序的保真度、信号强度和DNA消耗方面。

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.