Illumina MiSeq paired end sequencing; In Vitro Selection of DNA Molecules Favouring Intramolecular Circularization by DNA Ligase
In Vitro Selection of DNA Molecules Favouring Intramolecular Circularization by DNA Ligase
All DNA oligonucleotides were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). DNA sequence: ATCTC GACTA GTCAG N40 ATCCG AATGT CTCGG AT (N = A, T, G, C) (5' phosphate) The sample DNA was extracted by 10% denaturing polyacrylamide gel electrophoresis (dPAGE) or high-performance liquid chromatograph (HPLC). DNA molecules were concentrated by standard ethanol precipitation and purified by 10% dPAGE. The starting linear DNA library contains, in the 5' to 3' direction, a 5'-phosphate, a 5' 15-nt (nt: nucleotide) constant sequence element, a central 40-nt random region (N40), and a 3' 17-nt constant sequence element. The last sequence element is designed to a 6-base pair stem and a 5-nt loop, which positions the 3'-hydroxy terminus of the library in duplex form.