Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products
Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products
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DOI：
10.1006/abio.1999.4171
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发表时间：
1999-08-01
影响因子：
2.9
通讯作者：
Gjerde, DT
中科院分区：
文献类型：
--
作者：
Hecker, KH;Taylor, PD;Gjerde, DT
一种特殊形式的离子对反相高效液相色谱法在单核苷酸多态性（SNP）突变检测中获得了广泛的应用。该技术依赖于通过色谱分离部分变性的DNA异源双链体与同源双链体的温度调节异源双链体分析（TMHA）。在这里，我们证明了荧光标记是兼容的突变分析，通过这种形式的DNA色谱法，并提供了使用未标记的DNA片段的优势。TMHA野生型和突变等位基因的均匀标记产生与未标记片段相同的峰模式。然而，荧光标记增加保留时间，但不影响异源双链体和同源双链体的分辨率。它们增加了灵敏度并减少了分析所需的DNA量;例如，在这里提出的情况下，一个等位基因可以在另一个等位基因超过500倍的情况下被检测到。此外，仅在一条链上荧光标记的等位基因特异性野生型探针使得有可能选择性地监测特异性同源双链体和野生型/突变体异源双链体。这与内部同源双链标准品结合，与UV中记录的色谱图相比，大大降低了荧光色谱图的复杂性。这些简化的色谱图，其中在突变的存在下仅检测到内部同源双链标准品和标记的异源双链，极大地促进了突变等位基因的检测和鉴定。(C)北京：科学出版社.
A specialized form of ion-pair reversed-phase highperformance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wildtype and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes hom homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type/mutant heteroduplexes. This, in combination with an internal homoduplex standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles. (C) 1999 Academic Press.