Direct sequencing is the gold standard for genome haplotyping, while PCR is used for detection of limited number of genetic variations. However, these methods are not suitable for fast low-cost in-field testing of plant specimens. Here, we developed a two-color florescent nanosensor system (named here biplex deoxyribozyme nanomachine or bixDNM) that can haplotype dsDNA amplicons. The sensor is based on fluorescent binary deoxyribozyme embedded in a nanostructure containing together four DNA binding arms. Two allele specific sensors were tailored for producing signals at two different wavelengths (525 and 662 nM) only in the presence of fully matched analytes. Accurate haplotyping of seven barley samples was achieved. This is the first technique showing detected of both short (146 bp) and long (1348 bp) dsDNA amplicons with single nucleotide specificity. The study demonstrated bixDNM can become a foundation for future development of a plant haplotyping technology that can be used in field or at the low resource agricultural settings.

直接测序是基因组单倍型分析的金标准，而 PCR 用于检测有限数量的遗传变异。然而，这些方法不适用于植物标本的快速低成本现场测试。在这里，我们开发了一种双色荧光纳米传感器系统（此处命名为双链脱氧核酶纳米机器或 bixDNM），可以单倍型 dsDNA 扩增子。该传感器基于嵌入包含四个 DNA 结合臂的纳米结构中的荧光二元脱氧核酶。两个等位基因特异性传感器经过定制，仅在存在完全匹配的分析物时才可在两个不同波长（525 和 662 nM）下产生信号。实现了七个大麦样品的准确单倍型分析。这是第一项显示检测到具有单核苷酸特异性的短 (146 bp) 和长 (1348 bp) dsDNA 扩增子的技术。该研究表明 bixDNM 可以成为未来开发可用于田间或低资源农业环境的植物单倍型技术的基础。