Study On SELEX Screening Of Tetrodotoxin Aptamer And Its Application

In this article,the DNA aptamer that specially bind against tetrodotoxin with a high affinity was screened from a large random ssDNA library by SELEX screening technology.The application of DNA aptamer was preliminarily discussed,and it will lay a foundation for detection tetrodotoxin by aptamer in the further.Synthesized ssDNA library,a length of 78nt,is fixed sequence at both ends with 35 random sequence in the middle.Aiming to practice SELEX screening in the tetrodotoxin aptamer,acrylic beads activated by epoxy resin?Epoxy?were selected as solid phase separation media for coupling tetrodotoxin,combined with mutagenesis PCR technology.In the screening process,the PCR amplification conditions were optimized,the acrylic beads coupled with glycine was activated by the Epoxy and then doing anti-screening at the same time.The aptamer group selected from 13 round screening was cloned.Then the randomly chosen 19 clones were identified firstly and determined secondly.The primary structure and secondary structure of aptamer sequenced up to the standard was analyzed and the affinity was identified.Based on the the role of green fluorescent nucleic acid dye EvaGreen and aptamer-TTX complexes,TTX aptamer fluorescence detection methods were established.The SELEX screening results showed that the combination rate of ssDNA enriched library of each round with target molecule showed increasing tendency,and the absorbance value improved 4.0 times,from 0.235 of the first round to 0.936 of the 13 round.The conserved sequences of primary structure also revealed that 4 pairs of aptamers had identical sequences and 6 pairs aptamers?4 pairs of 6 pairs aptamers had identical sequences and there was only one or two different base in sequences of another 2 of aptamers.?This indicated that the aptamers with primary structure homology had been enriched,to some extent.The mimic diagram of aptamers structure exhibited that the secondary structure consisted mainly of Stem-loop structure,which suggested that these Stem-loops were the basis for aptamers binding to target protein.The experiments of aptamer binding to TTX showed that the combination rate of NO.A3 aptamer was maximum.The optimization reaction conditions results of No A3 aptamer and TTX showed that optimum pH of the combination was 7.5;the best time of dye combination was 10 min.Under the optimal conditions,the detection limit of the TTX was 10^-6mol/L.