Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons.
Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.
Tang
Zhiwen
Z
State Key Laboratory of Chemo/Biosensing and Chemometrics, Institute of Biological Technology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People's Republic of China.
Wang
Kemin
K
Tan
Weihong
W
Li
Jun
J
Liu
Lingfeng
L
Guo
Qiuping
Q
Meng
Xiangxian
X
Ma
Changbei
C
Huang
Shasheng
S
D011088:DNA Ligases / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D015336:Molecular Probe Techniques; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009696:Nucleic Acids / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012996:Solutions; D013696:Temperature; D013997:Time Factors
D015342:DNA Probes; D009696:Nucleic Acids; D012996:Solutions; D011088:DNA Ligases