Wilson R K, Chen C, Hood L
California Institute of Technology.
Biotechniques. 1990 Feb;8(2):184-9.
A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA.
本文描述了一种制备单链模板DNA的高通量方法，该方法适用于使用荧光标记化学进行序列分析。在此过程中，采用不对称聚合酶链反应直接从菌落或噬菌斑中扩增重组质粒或噬菌体DNA。使用位于测序引物退火位点5'端至少200个碱基对处的扩增引物，无需对不对称聚合酶链反应产物进行大量纯化。相反，单链产物DNA通过简单的异丙醇沉淀步骤进行纯化，然后使用荧光染料标记的寡核苷酸直接测序。该方法显著减少了模板制备所需的时间和劳动力，并通过提供更均匀的单链DNA产量改进了荧光DNA测序策略。
