Hairpin
Probes for Real-time Assay of Restriction Endonucleases
ZHANG Yong-You,LI Qing-Ge*,LIANG
Ji-Xuan,ZHU Yan-Bing
( The Key Laboratory of Cell Biology and Tumor Cell Engineering of Ministry
of Education,School of Life Sciences,Xiamen University,Xiamen 361005,China
)
Abstract New
fluorogenic probes for restriction endonucleases based on molecular beacons
were developed. These hairpin probes were single-stranded oligonucleotides that
had stem-and-loop structure and carried 5'- fluorophor moiety and 3'-quencher
moiety. Their stem sequences were designed as recognition sites for restriction
endonucleases and loop sequences were unrelated nucleotides.Upon cleavage by
endonuclease, these probes became fluorescent and thus could trace the enzyme
activity continuously. Two probes were designed for BglII and NcoI,
respectively, and each was labeled with a fluorophor of different color.The
results showed that the two probes could specifically assay for the
corresponding enzymes either individually or simultaneously in a real-time
mode. Considering the simplicity, quantification and high throughput,these
probes could be extended to other applications such as drug screening,
protein-nucleic acid interaction study and searching for small molecule DNA
cleavage agents.
Key words restriction endonuclease; hairpin probe;
real-time detection
*Corresponding
author:Tel,86-592-2187363;
e-mail,[email protected]