Molecular logic gates for DNA analysis: detection of rifampin resistance in M. tuberculosis DNA.
Molecular logic gates for DNA analysis: detection of rifampin resistance in M. tuberculosis DNA.
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DOI：
10.1002/anie.201203708
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发表时间：
2012-09-03
影响因子：
16.6
通讯作者：
Kolpashchikov, Dmitry M
中科院分区：
文献类型：
--
作者：
Cornett, Evan M;Campbell, Eleanor A;Gulenay, George;Peterson, Evan;Bhaskar, Neha;Kolpashchikov, Dmitry M
由DNA制成的分子逻辑门因其生物相容性、简单的设计及其分析和控制生物系统的能力而引起了人们的广泛关注。 [1]为了推动该领域的进一步发展，需要应用基于 DNA 的门来解决重大的生物问题。最近，我们表征了一组 DNA 逻辑门（YES、NOT、AND 和 OR），并通过设计 ANDNOT 和 XOR 运算展示了它们的连接性。[1g, h] 这些门使用 DNA 链与分子信标 (MB) 探针杂交来产生荧光输出。在这里，我们演示如何应用 DNA 逻辑门来解决一项重要的生物医学任务，即分析包含一组复杂突变的多个 DNA 序列。结核分枝杆菌 (Mtb) 感染全世界约 20 亿人，每年导致约 200 万人死亡。 [2]大约 10% 的患者感染了具有耐药性的 Mtb 菌株；这些菌株主要对抗生素利福平 (Rif) 和异烟肼具有耐药性。 [3]目前，迫切需要具有成本效益的诊断工具，可以检测临床样本中的 Mtb 并区分药物敏感和耐药 Mtb 菌株。[2-4] 存在多种检测方法来检测导致抗生素耐药性的突变。[5] Cepheid 的 Expert MTB/RIF 是最先进的商业化验之一，[5i-n] 利用实时 PCR (rtPCR) 和 MB 探针。[6] MB 探针首先由 Tyagi 和 Kramer 引入，[6a] 是茎环折叠寡核苷酸，其两端连接有荧光团和淬灭染料（图 1）。 MB 探针与互补 DNA 或 RNA 的杂交将探针转变为伸长形式，从而将荧光团与猝灭剂分离。由此产生的荧光增加可以被定量测量，这是MB探针在核酸实时检测中广泛应用的基础。 [6]在 Expert MTB/RIF 测定中，设计了 5 个 MB 探针来跨越细菌高度可变的 81-nt 核心。
Molecular logic gates made of DNA have attracted significant attention because of biocompatibility, simple design and their ability to analyze and control biological systems.[1] To fuel further development of the field, applications of DNA-based gates to solve significant biological problems are required. Recently we characterized a set of DNA logic gates (YES, NOT, AND, and OR) and demonstrated their connectivity by designing ANDNOT and XOR operations.[1g, h] The gates used hybridization of DNA strands with a molecular beacon (MB) probe to produce a fluorescent output. Here we demonstrate how DNA logic gates can be applied to solve an important biomedical task of analysis of multiple DNA sequences containing a complex set of mutations.Mycobacterium tuberculosis (Mtb) infects approximately 2 billion people all over the world and is responsible for about 2 million deaths each year.[2] Approximately 10% of all patients are infected by strains of Mtb that are drug-resistant; these strains are primarily resistant to antibiotics rifampin (Rif) and isoniazid.[3] Currently, there is an urgent need for costeffective diagnostic tools that can detect Mtb in clinical samples and differentiate between drug-susceptible and drug-resistant Mtb strains.[2-4] Several assays exist to detect mutations responsible for antibiotic resistance.[5] One of the most advanced commercial assays, Cepheid's Expert MTB/RIF,[5i-n] takes advantage of real-time PCR (rtPCR) and MB probes.[6] MB probes, first introduced by Tyagi and Kramer,[6a] are stem-loop folded oligonucleotides with fluorophore and quencher dyes attached at opposite ends (Fig. 1). Hybridization of an MB probe to a complementary DNA or RNA switches the probe to an elongated form, thus separating the fluorophore from the quencher. The resultant fluorescence increase can be quantitatively measured, which is the basis for the widespread application of MB probes in real-time detection of nucleic acids.[6] In the Expert MTB/RIF assay, five MB probes were designed to span the highly variable 81-nt core of the bacterial