DNA aptamer selection in vitro for determining ketamine by FluMag-SELEX.
- Author:
Mei-Qi SUN
;
Fang-Qi CAO
;
Xiao-Long HU
;
Yu-Rong ZHANG
;
Xin-Wei LU
;
Li-Bo ZENG
- Publication Type:Journal Article
- MeSH:
Aptamers, Nucleotide/metabolism*;
DNA;
DNA, Single-Stranded/genetics*;
In Vitro Techniques;
Ketamine/metabolism*;
Oligonucleotides;
SELEX Aptamer Technique/methods*
- From:
Journal of Forensic Medicine
2014;30(5):346-349
- CountryChina
- Language:Chinese
-
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.
