Perry D J
Department of Haematology, Haemophilia Centre and Haemostasis Unit, Royal Free Hospital School of Medicine, London, UK.
Methods Mol Med. 1999;31:49-54. doi: 10.1385/1-59259-248-1:49.
A novel approach to generating high-quality single-stranded DNA involves solid-phase sequencing. A biotin group is incorporated at the 5'-end of one of the amplification primers and as a result becomes incorporated into PCR product during the amplification reaction. The DNA can then be immobilized onto streptavidin-coated paramagnetic beads, simultaneously removing buffers, dNTPs, and unincorporated PCR primers. By alkali denaturation, the immobilized double-stranded DNA is converted into a single-stranded template, which can then be purified away from its complementary strand, avoiding any competition in the subsequent sequencing reaction. Although the protocol detailed in this chapter is designed to purify and sequence PCR products, the method is broadly applicable to the solid-phase sequencing of many other templates, e.g., plasmid DNA.
一种生成高质量单链DNA的新方法涉及固相测序。生物素基团被掺入其中一个扩增引物的5'端，因此在扩增反应过程中会掺入到PCR产物中。然后可以将DNA固定在链霉亲和素包被的顺磁性珠子上，同时去除缓冲液、dNTP和未掺入的PCR引物。通过碱变性，固定的双链DNA被转化为单链模板，然后可以将其与其互补链分离纯化，避免在后续测序反应中产生任何竞争。尽管本章详细介绍的方案旨在纯化和测序PCR产物，但该方法广泛适用于许多其他模板的固相测序，例如质粒DNA。
