Journal of Forensic Medicine

›› 2014, Vol. 30 ›› Issue (5): 346-349.DOI: 10.3969/j.issn.1004-5619.2014.05.007

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DNA Aptamer Selection in vitro for Determining Ketamine by FluMag-SELEX

SUN MEI-QI1,2, CAO FANG-QI3, HU XIAO-LONG2, ZHANG YU-RONG2, LU XIN-WEI1,2, ZENG LI-BO2   

  1. （1. State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China; 2. Shanghai Key Laboratory of Crime Scene Evidence, Key Laboratory of Forensic Evidence and Science Technology, Ministry of Public Security, Institute of Forensic Science, Shanghai Public Security Bureau, Shanghai 200083, China; 3. Shanghai Research Institute of Criminal Science and Technology, Shanghai 200083, China）
  • Online:2014-10-25 Published:2014-10-28

Abstract: Objective To select specific DNA aptamer for determining ketamine by FluMag-SELEX. Methods Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. Results Two ssDNA aptamers （Apt#4 and Apt#8） were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer’s structure. And loop and G-tetramer was the key of specific binding of ketamine. Conclusion FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.

Key words: molecular biology, ketamine, SELEX aptamer technique

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