Indirect purification method provides high yield and quality ssDNA sublibrary for potential aptamer selection.
The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin-strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020±210nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary.
Shenzhen Blood Center Institute of Transfusion Medicine, Shenzhen 518035, China. Electronic address: zyz200157@sohu.com.
Yinze
Y
Zhang
Shaanxi Blood Center, Xi'an 710061, China.
Hua
H
Xu
The Second Affiliated Hospital Guangzhou University of Chinese Medicine, Guangzhou 510105, China.
Huayou
H
Zhou
Shenzhen Blood Center Institute of Transfusion Medicine, Shenzhen 518035, China.
Fan
F
Wu
Shenzhen Blood Center Institute of Transfusion Medicine, Shenzhen 518035, China.
Yuqin
Y
Su
Shenzhen Blood Center Institute of Transfusion Medicine, Shenzhen 518035, China.
Yanlian
Y
Liang
Shenzhen Blood Center Institute of Transfusion Medicine, Shenzhen 518035, China.
Dan
D
Zhou
D001710:Biotin / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D052156:SELEX Aptamer Technique / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D004277:DNA, Single-Stranded; D001710:Biotin; D019809:Streptavidin
Aptamer; Asymmetric PCR; Biotin–streptavidin magnetic beads; Single-stranded DNA purification; Symmetric PCR