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Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix
Du, M; Flanagan, JH; Lin, BC; Ma, YF
关键词Capillary Electrophoresis Dna Mutation Dynamic Coating
刊名ELECTROPHORESIS
2003-09-01
DOI10.1002/elps.200305572
24期:18页:3147-3153
收录类别ISTP ; SCI
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences
类目[WOS]Biochemical Research Methods ; Chemistry, Analytical
研究领域[WOS]Biochemistry & Molecular Biology ; Chemistry
关键词[WOS]DOUBLE-STRANDED DNA ; GRADIENT GEL-ELECTROPHORESIS ; POINT MUTATIONS ; CONFORMATION POLYMORPHISM ; INTERCALATING DYES ; FRAGMENT ANALYSIS ; POLYACRYLAMIDE ; GENE ; BASES ; PURIFICATION
英文摘要The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.
语种英语
WOS记录号WOS:000185897100007
引用统计
被引频次:18[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/139245
专题中国科学院大连化学物理研究所
作者单位1.Univ Missouri, Dept Chem, Rolla, MO 65409 USA
2.Transgenom Inc, Omaha, NE USA
3.Chinese Acad Sci, Dalian Inst Chem Phys, Dalian, Peoples R China
推荐引用方式
GB/T 7714
Du, M,Flanagan, JH,Lin, BC,et al. Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix[J]. ELECTROPHORESIS,2003,24(18):3147-3153.
APA Du, M,Flanagan, JH,Lin, BC,&Ma, YF.(2003).Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix.ELECTROPHORESIS,24(18),3147-3153.
MLA Du, M,et al."Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix".ELECTROPHORESIS 24.18(2003):3147-3153.
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