Gapped-Duplex Structure to Label-Free Mismatch Detection of Pathogen DNA on Solid Substrate
Gold platforms have been commonly used for DNA genotype recognition. Conventional methods use vertically aligned single stranded (ss) DNA on gold as the probe, and then present the target ss-DNA to analyze hybridization. This method is time consuming, and the effectiveness is dependent on where the base pair mismatches occur. Our objective was to create a DNA-mismatch detection platform that is controllable, sensitive and label-free. Proposed is a gapped-duplex, label-free method that creates an enhanced capability of mismatch detection, as well as quicker response time. The probe DNA sequence of 40 base pairs specific to hsp 70 of human genotype of waterborne pathogen Cryptosporidium is placed between two thiolated DNA anchors of 28 base pairs each, making the total strand length 96 base pairs. The anchors are bound to the gold surface using thiol chemistry, and the alignment of DNA is controlled by the use of 6-mercapto-1-hexanol (MCH). This makes the desired sequence more accessible for hybridization of the target DNA strand. The DNA density, and hybridization were measured electrochemically using chronocoulometry (CC), and electrochemical impedance spectroscopy (EIS) respectively. The data collected shows that hybridization of matching target DNA for the proposed method can be seen after 15 minutes compared to at least 30 minutes for the conventional method. The overall hybridization response to matching target DNA, using a percent change, after 40 minutes was 355% for the stated method, versus 260% for the conventional. The proposed method has proven to overcome the major shortcomings associated with conventional DNA-DNA hybridization protocols. After evaluating this method for its hybridization efficiency, DNA mismatch discrimination, and probe density control, it was found to be a strong candidate for label free DNA mismatch detection that could be widely applied to DNA mutation and genetic disease detection.
Williams, Spencer, "Gapped-Duplex Structure to Label-Free Mismatch Detection of Pathogen DNA on Solid Substrate" (2014). Graduate Research Symposium. Paper 105.