Slightom J L, Sieu L C
Molecular Biology Unit 7242, Upjohn Company, Kalamazoo, MI 49007.
Biotechniques. 1992 Jul;13(1):94-105.
Primer-directed enzymatic sequencing has proven to be an efficient and effective method for sequencing various size double-stranded DNA templates. We previously developed a primer-directed sequencing procedure for using double-stranded cosmid (50 kb) DNAs as template. We are interested in using this method to directly sequence larger DNA templates. Towards this goal we applied this method to directly sequence an engineered gene that had been transferred and integrated into the 130-kb baculovirus genome. Both crudely prepared and CsCl gradient-banded baculovirus DNAs were tested and reasonable sequencing ladders were obtained for both types of DNA templates. As little as 3 micrograms of gradient-banded baculovirus DNA were found to be sufficient to obtain film exposure times similar to those observed for cosmid size templates, 24 to 48 h. Effectiveness of the described method was demonstrated by obtaining the complete sequence of the engineered respiratory syncytial virus chimeric FG gene (2.5 kb in length) directly from the recombinant baculovirus "Baculo-FG" genome. Thus, our results demonstrate first, that double-stranded DNA templates as large as 130 kb can be sequenced directly and second, that the nucleotide sequence of engineered genes integrated within the baculovirus genome can be determined without the use of any intermediate steps of procedures.
引物导向酶促测序已被证明是一种对各种大小的双链DNA模板进行测序的高效方法。我们之前开发了一种以双链黏粒（50 kb）DNA为模板的引物导向测序程序。我们有兴趣使用这种方法直接对更大的DNA模板进行测序。为了实现这一目标，我们应用该方法直接对一个已转移并整合到130 kb杆状病毒基因组中的工程基因进行测序。对粗制的和经氯化铯梯度分带的杆状病毒DNA都进行了测试，两种类型的DNA模板都获得了合理的测序梯带。发现仅3微克经梯度分带的杆状病毒DNA就足以获得与黏粒大小模板（24至48小时）相似的胶片曝光时间。通过直接从重组杆状病毒“Baculo-FG”基因组中获得工程化呼吸道合胞病毒嵌合FG基因（长度为2.5 kb）的完整序列，证明了所述方法的有效性。因此，我们的结果首先表明，长达130 kb的双链DNA模板可以直接测序，其次表明，无需任何中间步骤即可确定整合在杆状病毒基因组内的工程基因的核苷酸序列。
