Horai S
Nihon Rinsho. 1993 Sep;51(9):2258-63.
The use of magnet beads for the capture and purification of PCR product allows simple preparation of immobilized single-stranded template. This template is ideal for use in DNA direct sequencing. The end labeling of PCR product with biotin is achieved simply by using a biotinylated primer. Capture and purification are then accomplished by incubation with Streptavidin attached magnet beads and magnet separation. Once immobilized, the DNA is converted to a single-stranded template by elution with alkali and another magnetic separation. Immobilized single-stranded template prepared in this manner are suitable for manual and automated solid-phase DNA sequencing by the dideoxynucleotide chain termination method. A 100% single-stranded template is produced, without interference from primers, free nucleotides or the complementary strand. The solid phase approach provides reproducible sequencing results with high yields. The eluted complementary strand can also be sequenced after neutralization.
使用磁珠捕获和纯化PCR产物可实现固定化单链模板的简单制备。该模板非常适合用于DNA直接测序。通过使用生物素化引物可简单地实现PCR产物的生物素末端标记。然后通过与链霉亲和素包被的磁珠孵育并进行磁分离来完成捕获和纯化。固定后，通过用碱洗脱和另一次磁分离将DNA转化为单链模板。以这种方式制备的固定化单链模板适用于通过双脱氧核苷酸链终止法进行的手动和自动固相DNA测序。可产生100%的单链模板，不受引物、游离核苷酸或互补链的干扰。固相方法可提供高产率且可重复的测序结果。洗脱的互补链在中和后也可进行测序。
