Kaiser R J, MacKellar S L, Vinayak R S, Sanders J Z, Saavedra R A, Hood L E
Division of Biology, California Institute of Technology, Pasadena 91125.
Nucleic Acids Res. 1989 Aug 11;17(15):6087-102. doi: 10.1093/nar/17.15.6087.
Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.
基于荧光的自动化DNA序列分析为开展大规模测序项目提供了可能。定向策略，如特定引物定向测序方法（“基因步移”），在这类项目中应会证明很有用。本文描述了一项研究，该研究涉及将此方法与商用仪器（ABI 370A DNA测序仪）上的自动化荧光检测结合使用。这包括用于快速化学合成和纯化标记引物的程序、与商用分析软件兼容的引物序列设计，以及使用此类引物进行自动化DNA序列分析。一组四个荧光团标记的引物可在24小时内可靠合成，使用这组引物在另外24小时内可获得超过300个核苷酸的新分析序列。目前，扩大这些程序以充分利用测序仪的全部功能太慢且成本太高，不适合常规测序，因此特定引物的使用最适合填补使用随机克隆和测序策略产生的延伸序列中的缺口。
