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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.
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Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

机译：荧光能量转移染料标记的引物，用于DNA测序和分析。

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摘要

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.
机译：已经开发了利用荧光染料标记的DNA引物，这些引物利用了荧光能量转移（ET）来优化标记的吸收和发射特性。这些引物在5'端带有荧光素衍生物作为共同的供体，而其他荧光素和若丹明衍生物则作为引物连接到引物序列中修饰的胸苷残基上。通过在引物序列中放置修饰的胸腺嘧啶核苷来调节供体-受体间隔，可产生四个引物，所有引物在共同的激发波长（488 nm）处均具有强吸收性，并且荧光发射最大值为525、555、580和605纳米这些引物的ET效率在65％至97％的范围内，并且通过凝胶电泳显示出相似的电泳迁移率。通过氩离子激光激发，ET引物和由ET引物产生的DNA测序片段的荧光比相应的引物或用单一染料标记的片段的荧光高2至6倍。 ET引物的荧光强度更高，因此可以使用T7 DNA聚合酶通常需要的DNA模板的四分之一进行DNA测序。以单链M13mp18 DNA为模板，在商业测序仪上使用ET引物进行的典型测序反应可在前500个碱基中提供99.8％的准确度的DNA序列。 ET引物通常应用于开发其他多重DNA测序和分析方法。

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