Johnson A F, Lodhi M A, McCombie W R
Lita Annenberg Hazen Genome Center, Cold Spring Harbor Laboratory, New York 11724-0100, USA.
Anal Biochem. 1996 Oct 15;241(2):228-37. doi: 10.1006/abio.1996.0404.
A fluorescence-based, T7 (Sequenase) dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded binding protein is described. In this method, the circular, supercoiled DNA vector pUC19 is first linearized with a restriction enzyme to create a sequenceable template. Sequencing is then accomplished using three cycles of "denaturation," annealing, and extension/termination. Twenty-two of 33 hexamer strings tested in a controlled study produced acceptable sequence, with read lengths varying from the mid 300s to the low 400s and a base-calling accuracy of at least 97%. To test its potential utility in directed DNA sequencing, the protocol was then used to completely sequence both strands of pUC19. For this test project, a total of 28 hexamer strings was used with an overall successful priming rate of 75%. The current protocol appears to be sufficiently robust to be used in the finishing phase of a shotgun sequencing project and is amenable to semiautomation. Prospects for using the protocol for full-scale directed sequencing as well as for full automation are discussed.
描述了一种基于荧光的T7（测序酶）染料终止子法，用于对双链DNA进行测序，该方法使用由三个连续六聚体组成的序列作为引物，并结合单链结合蛋白。在该方法中，首先用限制性内切酶将环状超螺旋DNA载体pUC19线性化，以创建可测序模板。然后通过“变性”、退火和延伸/终止三个循环完成测序。在一项对照研究中测试的33个六聚体序列串中有22个产生了可接受的序列，读取长度从300多到400少不等，碱基识别准确率至少为97%。为了测试其在定向DNA测序中的潜在用途，该方案随后被用于对pUC19的两条链进行完全测序。对于这个测试项目，总共使用了28个六聚体序列串，总体成功引物率为75%。当前的方案似乎足够稳健，可用于鸟枪法测序项目的完成阶段，并且适合半自动化。还讨论了将该方案用于全面定向测序以及全自动化的前景。
