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. 1996 Jan;56(1):59-66. doi: 10.1016/0166-0934(95)01912-x.
Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.
本文描述了两种基于PCR的方法，用于获取未知序列的小环状RNA的克隆，且这些小环状RNA仅有微量可用。为避免对RNA序列做出任何假设，cDNA的合成以随机引物起始。然后使用一个引物对cDNA群体进行PCR扩增，该引物的序列存在于cDNA的两侧，这是因为它们是用随机六聚体获得的，然后将具有PCR引物序列的接头连接到它们的末端，或者因为cDNA是用一种寡核苷酸合成的，该寡核苷酸在其5'端包含PCR引物的序列，在其3'端有六个随机位置。这些方法仅需要约50 ng的纯化RNA模板。文中讨论了克隆假象出现的原因及避免这些假象的预防措施。
