Toneguzzo F, Glynn S, Levi E, Mjolsness S, Hayday A
EG&G Biomolecular, Natick, MA 01760.
Biotechniques. 1988 May;6(5):460-9.
Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.
介绍了使用通过改良的快速煮沸微量制备方案制备的超螺旋DNA作为模板进行可靠且准确的核苷酸序列分析的方法。该方法可在收获细菌后1小时内产生适用于测序的DNA模板。我们描述了在双脱氧核苷酸链终止反应中使用改良的T7 DNA聚合酶（测序酶）对超螺旋微量制备DNA进行测序的最佳反应条件。我们证明，在这些条件下，用微量制备DNA获得的测序数据与用CsCl纯化的超螺旋DNA获得的数据或使用单链DNA模板获得的数据没有区别。我们进一步表明，超螺旋DNA测序反应可以在市售的自动DNA测序系统上进行分析，该系统在电泳分离过程中检测32P标记的DNA。综上所述，这些进展代表了核苷酸序列分析过程中的重大改进。
