Ligase-based multiple DNA analysis by using an electrochemical sensor array.
We herein report an electrochemical biosensor for the sequence-specific detection of DNA with high discrimination ability for single-nucleotide polymorphisms (SNPs). This DNA sensor was constructed by a pair of flanking probes that "sandwiched" the target. A 16-electrode electrochemical sensor array was employed, each having one individual DNA capture probe immobilized at gold electrodes via gold-thiol chemistry. By coupling with a biotin-tagged detection probe, we were able to detect multiple DNA targets with a single array. In order to realize SNP detection, a ligase-based approach was employed. In this method, both the capture probe and the detection probe were in tandem upon being hybridized with the target. Importantly, we employed a ligase that specifically could ligate tandem sequences only in the absence of mismatches. As a result, when both probes were complementary to the target, they were ligated in the presence of the ligase, thus being retained at the surface during the subsequent stringent washing steps. In contrast, if there existed 1-base mismatch, which could be efficiently recognized by the ligase, the detection probe was not ligated and subsequently washed away. A conjugate of avidin-horseradish peroxidase was then attached to the biotin label at the end of the detection probe via the biotin-avidin bridge. We then electrochemically interrogated the electrical current for the peroxidase-catalyzed reduction of hydrogen peroxide. We demonstrated that the electrochemical signal for the wild-type DNA was significantly larger than that for the sequence harboring the SNP.
Wan
Ying
Y
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China.
Zhang
Jiong
J
Liu
Gang
G
Pan
Dun
D
Wang
Lihua
L
Song
Shiping
S
Fan
Chunhai
C
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D011088:DNA Ligases / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods
D004247:DNA; D011088:DNA Ligases