RESUMEN
The recognition that nucleic acids can be used as polymeric materials led to the blossoming of the field of DNA nanotechnology, with a broad range of applications in biotechnology, biosensors, diagnostics, and drug delivery. These applications require efficient methods to synthesize and chemically modify high molecular weight DNA. Here, we discuss terminal deoxynucleotidyl transferase (TdT)-catalyzed enzymatic polymerization (TcEP) as an alternative to conventional enzymatic and solid-phase DNA synthesis. We describe biochemical requirements for TcEP and provide step-by-step protocols to carry out TcEP in solution and from surfaces.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , ADN/biosíntesis , ADN/metabolismo , Peso Molecular , PolimerizacionRESUMEN
In this work, two electrochemical DNA sensors was developed based on the target induced structural switching of stem-loop probe (SLP) and surface initiated enzymatic polymerization (SIEP). Both of the electrochemical DNA sensors employed SLPs with the same sequence. However, one had a thiol label at its 3' terminal (the probe was named 3-SLP and the sensor was named 3-SLP-SENS) and the other at its 5' terminal (the probe was named 5-SLP and the sensor was named 5-SLP-SENS). In the initial state of the sensors, both of the probes adopted the stem-loop structure, which shielded the unlabeled terminals of capture probes from being approached. When the loop regions of the capture probes hybridized with the target DNA the conformation of the SLPs was changed to a rigid double-strand, as a result, the 5-SLP released a 3'-OH terminal for SIEP which could be catalyzed by terminal deoxynucleotidyl transferase (TdT). And the 3-SLP released a 5' phosphate terminal which is not suit for SIEP. Thus a signal probe was employed to hybridize with the 5 terminal of 3-SLP and provide a 3'-OH. Both of the sensors were then submitted to the TdT-mediated SIEP. By using biotinylated 2'-deoxyadenosine 5'-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA. Then avidin-horseradish peroxidases (Av-HRPs) were employed for specific binding to the biotin labels to produce electrochemical signals. The detection performances of two electrochemical DNA sensors were investigated and compared. It was demonstrated that though the 3-SLP-SENS employed extra signal probes, the background current was lower leading to a better detection limit. By taking advantage of SLP and SIEP, this 3-SLP-SENS has been able to detect as low as 0.1pM DNA targets with excellent differentiation ability for even single mismatches.