Wahlberg J, Holmberg A, Bergh S, Hultman T, Uhlén M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Electrophoresis. 1992 Aug;13(8):547-51. doi: 10.1002/elps.11501301112.
An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.
本文描述了一种使用机器人工作站进行固相DNA测序的综合方案，该方案包括DNA的磁分离以及通过电泳对测序产物进行分析，并自动检测荧光标记片段。该方法基于磁珠与链霉亲和素-生物素技术的结合，可用于基因组DNA和质粒DNA的测序。DNA模板通过聚合酶链反应（PCR）获得。文中描述了制备五个和十个固定样本的方案，分别产生10个和20个单链模板。磁纯化步骤在微量滴定板中进行，这允许采用一种综合方案，该方案涉及随后的自动引物退火和测序反应程序。在这里，通过使用荧光标记的测序引物对来自人类免疫缺陷病毒（HIV）感染患者的血液样本中的DNA和克隆的人类抗体DNA片段进行直接基因组测序来举例说明该程序。
