The Investigation Of Screening And Separation Method Of Affinity DNA Aptamer

Aptamer refers to the oligonucleotide fragments screened from the artificial construction of random single chain sequence, the screened oligonucleotide fragments can specially bind with various targets such as proteins, small molecules, organic ions and so on. It is usually a series of single-stranded nucleic acid molecule, which generally consist of 20 to 80 base pairs and is screened out by vitro screening system evolution ligands by exponential enrichment (systematic evolution of ligands by exponential enrichment, SELEX). Aptamer can recognise targets strictly and combine with targets in high affinity.At present,SELEX is the main method of screening aptamer and one kind of molecular evolutionary engineering technologies. The primary structure of nucleic acid molecular consist of a number of different combinations of nucleotide sequences. Artificial chemical synthesis techniques can be used to synthesize a length of designed different permutations of the nucleotide. There builds a nucleic acid library which contain all molecular with this length of all possible mutations. This can simulate the evolution of nature in the experimental conditions, to exert selective pressure, and to screen out the selected nucleic acid molecule finally.The key problem of SELEX is the separation of the combinative ssDNA with the target. The study focuses on the process of screening and separation problems in aptamer selection methods. This paper is divided into 3 parts, which separately investigate 3 methods of screening and separation of aptamer:Firstly, the method screens Classical swine fever virus monoclonal antibodies aptamer and HCG aptamer using SELEX technology, and uses colloidal gold as separation medium. After 12 rounds selection,polyacrylamide gel (PAGE) shows target oligonucleotide bands are missing. By ussing ultra filtration centrifuge tube as separation medium, we continued to screen HCG aptamer. Finally PAGE shows that 10th lotion still have DNA bands in 5th screening.Secondly, double-stranded DNA library method using nitrocellulose membrane (NC) as separation medium screening HCG aptamer.This method uses producted NC membrane-labeled antibody as a target and random double-stranded DNA library as the starting library to screen aptamer. After 1 round selection, polyacrylamide gel (PAGE) shows target oligonucleotide band. However, the balnk membrane wihch is used for comparison also had the same band of oligonucleotides.Thirdly, denaturation-renaturation method screens alkaline protease K aptamer. The method uses NC and PDVF membranes as separation medium and alkaline protease K as target. Polyacrylamide gel (PAGE) shows target oligonucleotide band, control and lotion does not have target oligonucleotide band. Thiol probe nucleic acid found that under the conditions that target nucleic acids and their PCR products are existent, after combined with the thiol probe, the amount of fluorescence of fluorescein maleimide can increase, the amount of fluorescence of control and lotion did not significantly increase.