Vende P, Le Gall G, Rasschaert D
INRA, unité de virologie et d'immunologie moléculaires, domaine de Vilvert, Jouy-en-Josas, France.
Vet Res. 1995;26(3):174-9.
A sequencing strategy based on the use of commercially available fluorescent-labeled universal primers for directly sequencing polymerase chain reaction (PCR) amplified material has been developed. The PCR reactions were performed with hybrid primers, specific to the viral sequence and possessing the sequence of the standard sequencing primers at their 5' end (M13 and reverse primers). These amplified fragments were sequenced by a classical dye-primer kit on an automated sequencer. As opposed to the use of fluorescent dideoxynucleotides, this sequencing method yielded accurate, high-grade sequences and had several advantages. First, the intensity of the sequencing peaks was much more homogeneous with the dye primer method. In addition, the problem of altered electrophoretic mobility, which may occur during the sequencing of custom-synthesised fluorescent primers, was avoided. This method was successfully reproduced using several different sets of chimeric primers. We believe that it is suitable for epidemiological studies conducted by nucleic sequence comparison, as in the case of rabbit haemorrhagic disease virus, as well as in other systems.
一种基于使用市售荧光标记通用引物直接对聚合酶链反应（PCR）扩增材料进行测序的策略已被开发出来。PCR反应使用杂交引物进行，这些引物对病毒序列具有特异性，并且在其5'端具有标准测序引物的序列（M13和反向引物）。这些扩增片段通过经典的染料引物试剂盒在自动测序仪上进行测序。与使用荧光双脱氧核苷酸不同，这种测序方法产生了准确、高质量的序列，并且具有几个优点。首先，染料引物法的测序峰强度更加均匀。此外，避免了在定制合成荧光引物测序过程中可能出现的电泳迁移率改变的问题。使用几组不同的嵌合引物成功重现了该方法。我们认为它适用于通过核酸序列比较进行的流行病学研究，如兔出血性疾病病毒的情况，以及其他系统。
