2-LABEL PEAK-HEIGHT ENCODED DNA SEQUENCING BY CAPILLARY GEL-ELECTROPHORESIS - 3 EXAMPLES
2-LABEL PEAK-HEIGHT ENCODED DNA SEQUENCING BY CAPILLARY GEL-ELECTROPHORESIS - 3 EXAMPLES
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DOI：
10.1093/nar/20.18.4873
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发表时间：
1992-09-25
影响因子：
14.9
通讯作者：
DOVICHI, NJ
中科院分区：
文献类型：
--
作者：
CHEN, DY;HARKE, HR;DOVICHI, NJ
我们报告了对塔博尔和理查森的峰高编码DNA测序技术的改进。如在原始方案中，测序反应使用具有锰而不是镁的修饰的T7聚合酶，以产生每个双脱氧核苷的非常均匀的掺入。为了提高测序准确度，在单独的测序反应中使用两种荧光标记的引物。例如，一种测序反应使用FAM标记的引物与双脱氧腺苷三磷酸和双脱氧胞嘧啶三磷酸;调节ddATP和ddCTP的浓度以产生片段相对强度的2：1变化。第二个测序反应使用塔姆拉标记的引物与双脱氧胸苷三磷酸和双脱氧胍三磷酸;调节ddTTP和ddGTP的浓度以产生片段相对强度的2：1变化。合并的反应产物通过毛细管凝胶电泳进行分离，并通过三种不同检测器系统之一进行鉴定。使用2：1的峰高比通常产生前350个碱基的97.5%的测序准确度; 3：1的峰高比将前400个碱基的准确度提高到99.5%。对于这些实验，毛细管电泳在200 V/cm的电场下进行;需要两到三个小时来分离长度高达400个核苷酸的测序片段。
We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.