Ultrasensitive near-IR fluorescence detection for capillary gel electrophoresis and DNA sequencing applications.
Ultrasensitive near-IR fluorescence detection for capillary gel electrophoresis and DNA sequencing applications.
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适用于毛细管凝胶电泳和 DNA 测序应用的超灵敏近红外荧光检测。
DOI：
10.1021/ac00115a010
复制
发表时间：
1995
影响因子：
7.4
通讯作者：
S. Soper
中科院分区：
文献类型：
--
作者：
D. Williams;S. Soper
本文介绍了用近红外荧光染料标记的寡核苷酸的电泳图，用毛细管凝胶电泳分离，并用超灵敏的近红外荧光检测系统检测。通用M13测序引物在5'端用含有异硫氰酸酯官能团的近红外染料标记。将近红外染料标记的测序引物与可见荧光素标记的引物在毛细管凝胶电泳中的柱上检测限进行比较，表明近红外病例的灵敏度有所提高。近红外染料标记引物的检出限为3.4 × 10(-20) mol（信噪比= 3），荧光素类似物的柱上检出限为1.5 × 10(-18) mol（信噪比= 3）。采用单线、单染色技术测定M13mp18模板的核苷酸碱基序列。在链延伸反应中使用的ddNTPs的摩尔浓度变化，达到4:2:1:0 （a:C:G:T）的比例，从而可以通过荧光强度测量来识别每个末端碱基。采用标准的Sanger双脱氧链终止技术、修饰的T7 DNA聚合酶和近红外染料标记的M13通用引物，从M13mp18模板制备测序阶梯。数据表明，采用4:2:1:0 （A:C:G:T）峰高识别方法确定的序列可靠，最多可达250个碱基。将M13mp18质粒的已知序列与使用该方法获得的序列进行比较，在4:2:1:0的比例下，碱基调用精度为84%。
Electropherograms of oligonucleotides labeled with near-IR fluorescent dyes, separated by capillary gel electrophoresis and detected using an ultrasensitive near-IR fluorescence detection system, are presented. A universal M13 sequencing primer was labeled on the 5' end with a near-IR dye containing an isothiocyanate functional group. Comparison of the on-column detection limits in capillary gel electrophoresis for the near-IR dye-labeled sequencing primer to those obtained for a visible fluorescein-labeled primer indicated improved sensitivity for the near-IR case. The detection limit was found to be 3.4 x 10(-20) mol (SNR = 3) for the near-IR dye-labeled primer, while the on-column detection limit for the fluorescein analog was 1.5 x 10(-18) mol (SNR = 3). The sequence of nucleotide bases in an M13mp18 template was determined using a single lane, single dye technique. The molar concentrations of the ddNTPs used during chain extension reactions were varied to achieve a ratio of 4:2:1:0 (A:C:G:T), which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the M13mp18 template using standard Sanger dideoxy chain-terminating techniques, the modified T7 DNA polymerase, and the near-IR dye-labeled M13 universal primer. The data indicated reliable sequence determination by the 4:2:1:0 (A:C:G:T) peak height identification method up to 250 bases from the annealing site. Comparison of the known sequence of the M13mp18 plasmid to that obtained using this protocol yielded a base-calling accuracy of 84% for the 4:2:1:0 ratio.
DOI：
10.1073/pnas.84.14.4767
发表时间：
1987-07-01
影响因子：
11.1
作者：
TABOR, S;RICHARDSON, CC
通讯作者：
RICHARDSON, CC
影响因子：
7.4
作者：
R. J. Williams;N. Narayanan;G. A. Casay;M. Lipowska;L. Strekowski,;G. Patonay;J. M. Peralta;V. Tsang
通讯作者：
R. J. Williams;N. Narayanan;G. A. Casay;M. Lipowska;L. Strekowski,;G. Patonay;J. M. Peralta;V. Tsang
影响因子：
7.4
作者：
MATHIES, RA;PECK, K;STRYER, L
通讯作者：
STRYER, L