Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario M1C 1A4, Canada.
Analyst. 2013 Jun 21;138(12):3538-43. doi: 10.1039/c3an36810f. Epub 2013 May 13.
High throughput DNA basepair mismatch detection is an ultimate goal for earlier and point-of-care diagnostics. However, the size of a target sequence on single nucleotide mismatch detection will critically impact the design of sensors in future. To study the potential impact of target size, the probe and target strands of unequal size were hybridized in the absence and presence of single nucleotide mismatches along the sequence. After hybridization, the shorter target sequences form overhangs in the probe strand while longer target sequences form overhangs in the complementary strand. The resulting double stranded DNA hybrids were printed on gold surfaces and the electrochemical response of the films was studied by scanning electrochemical microscopy without signal amplification and label. The redox mediator, Fe(CN)(6), experiences lower repulsion in the vicinity of mismatch containing ds-DNA films, which ultimately manifests into higher feedback current regardless of the size and hybridization position of the complementary strands. Kinetic rate constants monitored right above the ds-DNA films show k(0) = 4.5 ± 0.1 × 10(-5) cm s(-1) for the short sequence hybridized at the upper portion of the probe while k(0) = 4.1 ± 0.2 × 10(-5) cm s(-1) for longer complementary strands which has only top overhang. It suggests that hybridization position is important for mismatch detection in short complementary stands. However, in longer complementary strands, mismatches are easily detectable in the absence of bottom overhangs.
高通量 DNA 碱基错配检测是早期和即时诊断的最终目标。然而，在单核苷酸错配检测中，目标序列的大小将对未来传感器的设计产生至关重要的影响。为了研究目标大小的潜在影响，在不存在和存在单核苷酸错配的情况下，将不等大小的探针和靶链进行杂交。杂交后，较短的靶序列在探针链上形成突出，而较长的靶序列在互补链上形成突出。所得双链 DNA 杂交体被打印在金表面上，并且在没有信号放大和标记的情况下通过扫描电化学显微镜研究了薄膜的电化学响应。氧化还原介体 Fe(CN)(6) 在含有错配的 ds-DNA 薄膜附近受到的排斥力较低，最终无论互补链的大小和杂交位置如何，都会产生更高的反馈电流。在 ds-DNA 薄膜上方直接监测的动力学速率常数显示，短序列在探针上部杂交时的 k(0) = 4.5 ± 0.1 × 10(-5) cm s(-1)，而较长的互补链只有顶部突出时的 k(0) = 4.1 ± 0.2 × 10(-5) cm s(-1)。这表明杂交位置对于短互补链中的错配检测很重要。然而，在较长的互补链中，即使没有底部突出，错配也很容易检测到。
