Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.
Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.
复制标题
DOI：
10.1093/nar/gkw028
复制
发表时间：
2016-05-05
影响因子：
14.9
通讯作者：
Brown T
中科院分区：
文献类型：
--
作者：
Ren X;El-Sagheer AH;Brown T
设计了一种空间要求不高的dTTP叠氮类似物(AHP DUTP)，其碱基上带有烷基链和乙炔基键，并通过引物延伸、逆转录和聚合酶链式反应(PCR)将其整合到DNA中。叠氮修饰的523个碱基的聚合酶链式反应扩增子用AHP DU取代了全部335个胸腺嘧啶，是模板的完美拷贝。用AHP DU取代胸腺嘧啶核苷增加了双链稳定性，这在一定程度上解释了叠氮修饰的三磷酸的高掺入效率。聚合酶链式反应产物经λ-核酸内切酶消化、链霉亲和素磁珠分离后，可方便地制备叠氮化单链DNA。利用双环[6.1.0]-非-4-炔(BCN)官能化染料，通过菌株促进的炔-叠氮环加成反应(SPAAC)，实现了单链和双链DNA的高效荧光标记。这表明，必须仔细控制标记的程度，以获得最佳的荧光，避免荧光猝灭。双色探针是在单管荧光标记反应中获得的；改变两种染料的比例提供了一种简单的方法来制备具有独特荧光特征的DNA探针。AHP dUTP是一种多功能的可点击核苷酸，在生物和纳米技术中具有潜在的广泛应用，包括单分子研究和通过SELEX合成修饰的适体文库。
A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.