Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, CZ-16000 Prague 6, Czech Republic.
Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007 Tarragona, Spain.
Anal Chem. 2023 Aug 29;95(34):12586-12589. doi: 10.1021/acs.analchem.3c02023. Epub 2023 Aug 14.
The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.
先前报道的正交多潜能氧化还原编码所有四个 DNA 碱基的方法仅允许分析短 DNA 片段的相对核苷酸组成。在这里，我们提出了两种方法来归一化电化学读数，以方便确定总核苷酸组成。第一种方法基于 DNA 引物中是否存在 7-脱氮-2'-脱氧鸟苷内标。在两次平行分析和减去电化学信号强度后，确定了 DNA 的精确组成。第二种方法利用了 5'- 二茂铁修饰的引物，其中第五个正交氧化还原标记作为信号归一化的参考，从而可以在单次读取中进行准确的电化学序列分析。这两种方法都使用了各种序列进行了测试，并且使用内标或参考标记对获得的伏安信号进行了归一化，并证明与实际核苷酸组成完全一致，突出了针对 DNA 序列分析的潜力。
