White D W, Drossman H, Drayna D
Genomyx Corporation, South San Francisco, CA 94080.
Biotechniques. 1992 Aug;13(2):232-7.
A method for the preparation of homogeneous, single-stranded polydeoxynucleotides of desired length up to 800 bases is described. The procedure entails 1) generation of double-stranded DNA of desired length by PCR using a pair of primers of which one is biotinylated and the other is either unlabeled or fluorescently labeled, 2) isolation of PCR products by agarose slab gel electrophoresis, 3) recovery of desired product by electroelution, 4) binding of the product to streptavidin-coated magnetic beads and is followed by 5) duplex denaturation and removal of the unbound single strand that is either unlabeled or fluorescently labeled. Final product characteristics were determined by capillary gel electrophoresis with fluorescence detection. Up to microgram quantities of homogeneous single-stranded DNA of a desired length were obtained. These can be used as single-stranded size standards in capillary gel electrophoresis experiments as well as in other techniques requiring such standards.
本文描述了一种制备长度达800个碱基的均一、单链多脱氧核苷酸的方法。该方法包括：1）使用一对引物通过聚合酶链式反应（PCR）生成所需长度的双链DNA，其中一个引物被生物素化，另一个引物未标记或被荧光标记；2）通过琼脂糖平板凝胶电泳分离PCR产物；3）通过电洗脱回收所需产物；4）将产物与链霉亲和素包被的磁珠结合，随后进行5）双链变性并去除未结合的未标记或荧光标记的单链。最终产物特性通过带荧光检测的毛细管凝胶电泳确定。可获得微克量的所需长度的均一单链DNA。这些可作为毛细管凝胶电泳实验以及其他需要此类标准的技术中的单链大小标准。
