起始模板浓度对单链核苷酸适配体次级文库制备的影响

Effect of initial template concentration on the preparation of ssDNA sublibrary

目的制备高纯度高浓度的单链核苷酸(ss DNA)适配体次级文库。方法使用生物素标记的上、下游引物做PCR扩增制备双链DNA(ds DNA);以ds DNA为模板,使用大量无生素标记的上游引物及少量生物素标记的下游引物行不对称PCR扩增制备ss DNA;使用链霉亲和素磁珠去除ss DNA扩增产物中的ds DNA及其他副产物。结果起始模板浓度在1.13×(105-107)拷贝时,链霉亲和素磁珠去除ss DNA扩增产物中的ds DNA及其他副产品的效果较好;起始模板浓度≥1.13×108拷贝,则仍有较多副产品残留在纯化后的ss DNA产物中。结论起始模板浓度控制在1.13×105-1.13×107拷贝,应用本方法可获得纯度、产量均较高的ss DNA次级文库。

Objective To generate a high yield and quality ssDNA sub-library. Methods ds DNA were amplified using biotinylated primers to provide enough templates for asymmetric PCR. Asymmetric PCR was carried out with excess nonbiotinylated forward primer and limited biotinylated reverse primer to generate ss DNA. dsDNA and by-products were eliminated via streptavidin magnetic beads. Results The contaminants of dsDNA and by-products could be eliminated effectively by streptavidin magnetic beads when the initial template concentration was 1. 13 × 105-1. 13 × 107. However,when the initial template concentration was more than 1. 13 × 108,more by-product residues were found in the purified ss DNA products.Conclusion This method could provide a high yield and quality ss DNA sub-library by controlling the initial template concentration between 1. 13 × 105-1. 13 × 107.