Navarro B, Daròs J A, Flores R
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Spain.
J Virol Methods. 1998 Jul;73(1):1-9. doi: 10.1016/s0166-0934(98)00042-1.
A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.
本文描述了一种用于克隆小环状RNA的通用方案，该方案仅需极少量（约50 ng）未知序列的模板。两条cDNA链分别通过一个26聚体引物合成，该引物的3'端六个位置完全简并，分别在逆转录酶和DNA聚合酶催化的两个连续反应中使用。然后使用与前一个引物非简并序列相同的20聚体引物对cDNA进行PCR扩增，克隆并测序。这些信息可用于合成一对或多对特异性相邻引物，通过文中所述的方案获得全长cDNA克隆。
