Rapid sizing of short tandem repeat alleles using capillary array electrophoresis and energy-transfer fluorescent primers.
Rapid sizing of short tandem repeat alleles using capillary array electrophoresis and energy-transfer fluorescent primers.
复制标题
使用毛细管阵列电泳和能量转移荧光引物快速测定短串联重复等位基因的大小。
作者：
Y. Wang;J. Ju;B. A. Carpenter;J. Atherton;G. Sensabaugh;R. Mathies
使用毛细管阵列电泳和能量转移荧光染料标记的聚合酶链反应引物对短串联重复序列（STR）多态性HUMTHO 1进行基因分型。通过使用在5'端用一种荧光素标记并且在第15个（修饰的）碱基位置用另一种荧光素标记的引物扩增靶等位基因，以产生发绿色荧光的片段（λ max = 525 nm）。将未知等位基因与由具有6、7、8和9个四碱基对重复的等位基因组成的标准梯状物一起进行电泳分离，其中每个用能量转移引物扩增，所述能量转移引物在5'末端具有供体荧光素，在第7个（修饰的）碱基的位置具有罗丹明受体，以产生发红色荧光（> 590 nm）的标准片段。在填充有由0.8%羟乙基纤维素加1 μ M 9-氨基吖啶组成的可替换筛分基质的中空熔融石英毛细管阵列上进行分离以提高分辨率。在488 nm处激发标记的DNA片段，并用双色共聚焦荧光扫描仪检测荧光。分离在不到20分钟内完成，并且允许以小于0.4个碱基对的平均绝对误差或准确度和大约0.5个碱基对的平均标准偏差进行大小测定，而不校正荧光通道之间的迁移率偏移和串扰。本研究建立了利用毛细管阵列电泳对双链DNA片段进行高速、高通量STR分型的可行性。
Genetic typing of the short tandem repeat (STR) polymorphism HUMTHO1 has been performed using capillary array electrophoresis and energy-transfer fluorescent dye-labeled polymerase chain reaction primers. Target alleles were amplified by use of primers labeled with one fluorescein at the 5' end and another fluorescein at the position of the 15th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). Unknown alleles were electrophoretically separated together with a standard ladder made up of alleles having 6, 7, 8, and 9 four-base pair repeats, each of which was amplified with an energy-transfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standard fragments fluorescing in the red (> 590 nm). Separations were performed on arrays of hollow fused-silica capillaries filled with a replaceable sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 microM 9-aminoacridine to enhance the resolution. The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations are complete in less than 20 min and allow sizing with an average absolute error or accuracy of less than 0.4 base pair and an average standard deviation of approximately 0.5 base pair with no correction for mobility shift and cross-talk between the fluorescence channels. This work establishes the feasibility of high-speed, high-throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis.
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