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首页> 外文期刊>Journal of Molecular Biology Research >Time-Resolved F?rster Resonance Energy Transfer Analysis of Single-Nucleotide Polymorphisms: Towards Molecular Typing of Genes on Non-Purified and Non-PCR-Amplified DNA
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Time-Resolved F?rster Resonance Energy Transfer Analysis of Single-Nucleotide Polymorphisms: Towards Molecular Typing of Genes on Non-Purified and Non-PCR-Amplified DNA

机译：单核苷酸多态性的时间分辨弗斯特共振能量转移分析：未纯化和非PCR扩增的DNA基因的分子分型

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Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labeling the opposite ends of gene-specific oligonucleotide probes is a powerful tool to detect DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, time-correlated single-photon counting. Recently, we probed by such technique the highly polymorphic DQB1 human gene. Namely, by using a single oligonucleotide probe and acting on non-amplified DNA samples contained in untreated cell extracts, we demonstrated the ability of pursuing unambiguous recognition of subjects bearing the homozygous DQB1-0201 genotype by exploiting the subtle, yet statistically significant, structural differences between the duplex formed by the probe with DQB1-0201 on the one end and duplexes formed with any of the other alleles, on the other end. The relevance of homozygous DQB1-0201 genotype recognition reseeds in the fact that the latter is overexpressed in subjects affected by insulin-dependent diabetes mellitus in north-eastern Italy. In this article we review our preceding achievements and report on additional in-vitro experiments aimed at characterizing the duplexes obtained by annealing of the DQB1 allelic variants with a second oligonucleotide probe, with the final scope to achieve full genotyping of DQB1 on raw DNA samples by means of cross-combination of the FRET responses of both probes.
机译：标记基因特异性寡核苷酸探针相对两端的生色团之间的荧光共振能量转移（FRET）效率的定量评估是检测具有单核苷酸分辨率的DNA多态性的强大工具。通过应用时间分辨荧光分析方法，时间相关的单光子计数，可以最方便地量化FRET效率。最近，我们通过这种技术探索了高度多态的DQB1人类基因。即，通过使用单个寡核苷酸探针并作用于未经处理的细胞提取物中包含的未扩增的DNA样品，我们证明了通过利用细微但在统计学上显着的结构差异来追求具有纯合DQB1-0201基因型的受试者的明确识别的能力在一端具有DQB1-0201的探针形成的双链体与另一端与其他任何等位基因形成的双链体之间。纯合DQB1-0201基因型识别的相关性在于，后者在意大利东北部受胰岛素依赖型糖尿病影响的受试者中过表达。在本文中，我们回顾了我们先前的成就并报告了其他体外实验，这些实验旨在表征通过使用第二个寡核苷酸探针对DQB1等位基因变体进行退火而获得的双链体，最终范围是通过两种探针的FRET反应交叉组合的方法。

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