Flinders Centre for Nanoscale Science and Technology, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia 5042, Australia.
Flinders Centre for Nanoscale Science and Technology, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia 5042, Australia; Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, VIC 3010, Australia.
Anal Chim Acta. 2017 Feb 15;954:121-128. doi: 10.1016/j.aca.2016.12.011. Epub 2016 Dec 30.
Single nucleotide polymorphism (SNP) genotyping is an important aspect in understanding genetic variations. Here, we discriminate SNPs using toe-hold mediated displacement reactions. The biological target is an 80 nucleotide long double-stranded-DNA from the mtDNA HV1 region, associated with maternal ancestry. This target has been specially designed with a pendant toehold and a cationic fluorophore, ATTO 647N, as a reporter, produced in a polymerase chain reaction. Rates of reaction for the toehold-polymerase chain reaction products (TPPs) with their corresponding complementary displacing sequences, labelled with a Black Hole Quencher 1, followed the order TPP-Cytosine > TPP-Thymine > TPP-Adenine ≥ TPP-Guanine. Non-complementary rates were the slowest with mismatches involving cytosine. These reactions, operating in a static/or contact mode, gave averaged readouts between SNPs within 15 min (with 80-90% quenching), compared to 25-30 min in previous studies involving fluorescence resonance energy transfer. Addition of an intercalating agent, ethidium bromide, retarded the rate of reaction in which cytosine was involved, presumably through stabilization of the base pairing, which resulted in markedly improved discrimination of cytosine containing SNPs.
单核苷酸多态性 (SNP) 基因分型是理解遗传变异的一个重要方面。在这里，我们使用固着介导的置换反应来区分 SNP。生物靶标是来自 mtDNA HV1 区域的 80 个核苷酸长的双链 DNA，与母系祖先有关。该靶标经过专门设计，带有一个悬垂固着点和一个阳离子荧光团 ATTO 647N，作为报告分子，通过聚合酶链反应产生。带有互补置换序列的固着聚合酶链反应产物 (TPP) 的反应速率，用 Black Hole Quencher 1 标记，遵循 TPP-胞嘧啶 > TPP-胸腺嘧啶 > TPP-腺嘌呤 ≥ TPP-鸟嘌呤的顺序。非互补反应速率最慢，涉及胞嘧啶的错配最慢。这些反应以静态/接触模式进行，与以前涉及荧光共振能量转移的研究相比，在 15 分钟内（80-90%猝灭）对 SNP 进行了平均读数，而在以前的研究中需要 25-30 分钟。加入嵌入剂溴化乙锭会减缓涉及胞嘧啶的反应速率，这可能是通过稳定碱基配对来实现的，从而显著提高了含胞嘧啶 SNP 的区分能力。
