[DNA aptamer selection in vitro for determining ketamine by FluMag-SELEX].
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.


CONCLUSIONS
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.
Mei-Qi
MQ
Sun
Fang-Qi
FQ
Cao
Xiao-Long
XL
Hu
Yu-Rong
YR
Zhang
Xin-Wei
XW
Lu
Li-Bo
LB
Zeng
D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D004277:DNA, Single-Stranded / Q000235:genetics*; D066298:In Vitro Techniques; D007649:Ketamine / Q000378:metabolism*; D009841:Oligonucleotides; D052156:SELEX Aptamer Technique / Q000295:instrumentation / Q000379:methods*
D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D007649:Ketamine; D004247:DNA