Isolation of single-stranded DNA using denaturing DNA chromatography
Isolation of single-stranded DNA using denaturing DNA chromatography
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DOI：
10.1006/abio.2000.4669
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发表时间：
2000-08-15
影响因子：
2.9
通讯作者：
Hornby, DP
中科院分区：
文献类型：
--
作者：
Dickman, M;Hornby, DP
单链DNA（single-strandedDNA，ssDNA）是分子生物学中非常重要的试剂。ssDNA的纯化和分离是许多分析分子生物学程序中的关键步骤，包括链特异性杂交（1）、ssDNA适体的体外选择（2，3）、核苷酸测序（4）和使用质谱法的DNA分子量分析（5）。先前已经使用各种方法从双链（ds）PCR产物获得ssDNA（6-13）。重要的是，大多数技术需要一个以上的步骤来从dsPCR产物中分离纯化的ssDNA。在这里，我们报告了一种技术，使我们能够产生单链DNA直接从双链PCR产物产生的使用“标记引物”。“标签”可以是任何疏水部分：在以下程序中使用生物素基团和荧光素两者。从dsPCR产物中直接获得ssDNA的速度显著提高（即小于15分钟），同时从PCR产物中产生特定大小的高度纯化的ssDNA，并允许快速分离荧光标记的ssDNA。该方法使用DNA Wave片段分析系统，该系统结合了使用DNA Sep技术的变性反相离子对高效液相色谱（RP-IP DHPLC）1（14）。结果和讨论。双链DNA
Single-stranded DNA (ssDNA) is an extremely important reagent in molecular biology. The purification and isolation of ssDNA is a key step in numerous analytical molecular biology procedures, including strand-specific hybridization (1), the in vitro selection of ssDNA aptamers (2, 3), nucleotide sequencing (4), and molecular weight analysis of DNA using mass spectrometry (5). Various methods have been used previously to obtain ssDNA from double-stranded (ds) PCR products (6–13). Importantly most of the techniques require more than one step to isolate the purified ssDNA from dsPCR products. Here we report a technique that allows us to generate single-stranded DNA directly from doublestranded PCR products generated using a “tagged primer.” The “tag” can be any hydrophobic moiety: both a biotin group and fluorescein were used in the following procedure. A dramatic increase in the speed in which ssDNA can be obtained directly from dsPCR products (ie, less than 15 min), which simultaneously generates highly purified ssDNA of a specific size from the PCR products and also allows the rapid isolation of fluorescently labeled ssDNA. This method uses the DNA Wave fragment analysis system that incorporates denaturing reverse-phase ion pair high-performance liquid chromatography (RP-IP DHPLC) 1 using DNA Sep technology (14).Results and discussion. Double-stranded DNA