从病毒RNA模板合成长链cDNA。
Synthesis of long cDNA from viral RNA template.
作者信息
Lenstra J A, de Groot R J, Jacobs L, Kusters J G, Niesters H G, van der Zeijst B A
机构信息
Institute of Infectious Diseases and Immunology, Veterinary Faculty, State University of Utrecht, The Netherlands.
出版信息
Gene Anal Tech. 1988 May-Jun;5(3):57-61. doi: 10.1016/0735-0651(88)90017-9.
Abstract
Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.
摘要
从病毒RNA模板制备长且可靠的cDNA的方法已得到优化。病毒RNA模板的变性条件最为关键。对于第一链DNA的合成，引物浓度和核糖核酸酶抑制剂的存在很重要。在第二链合成过程中，发现孵育温度对转录本长度有影响。将我们优化的条件应用于冠状病毒基因组RNA作为模板，得到了插入片段大小在0.5 - 5 kb范围内的cDNA文库，无需单独进行cDNA大小选择。此外，还描述了一种简便的乙醇沉淀变体以及在中性琼脂糖凝胶上分析单链DNA的方法。
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