Highly sensitive DNA detection and point mutation identification: an electrochemical approach based on the combined use of ligase and reverse molecular beacon.
A novel strategy is described for highly sensitive DNA detection and point mutation identification based on the combination of reverse molecular beacon with DNA ligase. A 5'-phosphoryl and 3'-ferrocene terminated DNA sequence is used as detection probe, which may be ligated to capture DNA immobilized on an electrode surface in the presence of a target DNA strand that is complementary to the ends of each DNA, since this allows formation of a nicked, double-stranded DNA. The ligation product may form a hairpin structure after the removal of target DNA. By this method, target DNA can be determined in the range from 3.4 x 10(-12) to 1.4 x 10(-7) M with a detection limit of 1.0 x 10(-12) M. In contrast to existing methods based on the conformation change of redox-labeled oligonucleotides, the proposed strategy offers several substantial advantages: first, the background peak current is eliminated as the ferrocene (Fc)-tagged oligonucleotide probe is specifically ligated to capture DNA; second, a "signal-on" mechanism makes the current intensity increase with increasing target DNA concentration; third, improved current signal is obtained due to the formation of the hairpin structure of ligation products. Additionally, the present system exhibits excellent capability to discriminate mutant target sequences from fully complementary target sequences.
Wu
Zai-Sheng
ZS
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, PR China.
Jiang
Jian-Hui
JH
Shen
Guo-Li
GL
Yu
Ru-Qin
RQ
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D011088:DNA Ligases / Q000737:chemistry*; D004563:Electrochemistry; D004566:Electrodes; D005296:Ferrous Compounds / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D000075163:Metallocenes; D009693:Nucleic Acid Hybridization / Q000235:genetics; D010472:Perchlorates / Q000737:chemistry; D017354:Point Mutation / Q000235:genetics*; D012680:Sensitivity and Specificity; D017670:Sodium Compounds / Q000737:chemistry
D005296:Ferrous Compounds; D000075163:Metallocenes; D010472:Perchlorates; D017670:Sodium Compounds; D006046:Gold; D004247:DNA; C031068:sodium perchlorate; D011088:DNA Ligases; C120640:DNA ligase (NAD); C004998:ferrocene