Rezaian M A
CSIRO Plant Industry Horticulture Unit, Adelaide Laboratory, PO Box 350, Glen Osmond, SA 5064, Australia.
Curr Issues Mol Biol. 1999;1(1-2):13-20.
Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact-length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules.
类病毒是小型自主复制的RNA，与植物的其他亚病毒环状单链RNA具有共同的结构特征。类病毒和其他环状单链RNA可以通过基于PCR的程序，利用一组简单的反应在体外合成。从类病毒的一个所需区域选择两个首尾相接的引物，一个用于合成第一链cDNA，另一个用于生成第二链DNA。第二个引物在其5'端含有一个18个核苷酸的T7启动子，其选择方式使得转录起始位点的G核苷酸在类病毒中也代表G。连接的逆转录PCR产生由类病毒序列和T7启动子组成的线性双链DNA。PCR产物的连续转录允许合成精确长度的线性类病毒RNA，在将5'三磷酸酶修饰为单磷酸酶后，该RNA可通过T4 RNA连接酶环化。此程序可产生真实的类病毒分子，无需构建和克隆串联重复形式的DNA进行感染性测试。它还允许基于PCR对环状RNA进行操作，从而极大地简化了类病毒分子的结构-功能分析。
