DIRECT SEQUENCE-ANALYSIS OF AMPLIFIED DENGUE VIRUS GENOMIC RNA FROM CULTURED-CELLS, MOSQUITOS AND MOUSE-BRAIN
DIRECT SEQUENCE-ANALYSIS OF AMPLIFIED DENGUE VIRUS GENOMIC RNA FROM CULTURED-CELLS, MOSQUITOS AND MOUSE-BRAIN
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DOI：
10.1016/0166-0934(92)90029-d
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发表时间：
1992-06-01
影响因子：
3.1
通讯作者：
DALGARNO, L
中科院分区：
文献类型：
--
作者：
LEE, E;NESTOROWICZ, A;DALGARNO, L
本发明描述了一种用于直接序列分析感染组织中登革病毒基因组RNA的选定区域的方法。 使用特异性引物，将总高分子量感染细胞RNA逆转录为单链（ss）互补DNA，使用聚合酶链式反应（PCR）扩增，并使用λ外切核酸酶消化PCR产物的一条链后获得的ssDNA测序（R.G. Higuchi和H. Ochman，Nucleic Acids Research，17，5865，1989）。 使用来自少量（10（5））培养的蚊子或猴肾细胞、来自1 mg感染的小鼠脑和来自1/300感染的Toxorhynchites amboinensis蚊子的RNA获得两种登革-3病毒分离物的包膜蛋白基因的序列数据。 独立测定表明，逆转录或PCR过程中发生的错误在扩增DNA的序列中并没有显着表现出来。 该方法不依赖于病毒的广泛传代或大规模生长来产生用于测序的材料，因此提供了获得未适应的登革病毒分离株的序列数据的手段。
A method is described for direct sequence analysis of selected regions of dengue virus genomic RNA in infected tissues. Using specific primers, total high-molecular-weight infected-cell RNA is reverse transcribed to single-stranded (ss) complementary DNA, amplified using the polymerase chain reaction (PCR) and sequenced using ssDNA obtained after lambda exonuclease digestion of one strand of the PCR product (R.G. Higuchi and H. Ochman, Nucleic Acids Research, 17, 5865, 1989). Sequence data for the envelope protein gene of two dengue-3 virus isolates were obtained using RNA from small numbers (10(5)) of cultured mosquito or monkey kidney cells, from one mg of infected mouse brain and from 1/300th of an infected Toxorhynchites amboinensis mosquito. Independent determinations showed that errors occurring during reverse transcription or PCR were not represented to a significant degree in the sequence of the amplified DNA. The method does not depend on extensive passaging of virus or large-scale growth to generate material for sequencing and therefore provides a means of obtaining sequence data for unadapted dengue virus isolates.