Bertling W M, Beier F, Reichenberger E
Paul-Ehrlich-Institute, Unit of Molecular Pathology, Langen, Germany.
PCR Methods Appl. 1993 Oct;3(2):95-9. doi: 10.1101/gr.3.2.95.
We established a novel way to clone 5' ends of unknown length and sequence of individual cDNAs. T4 DNA ligase is employed to ligate an annealed duplex of complementary primers, one of them with a 4-nucleotide-long randomized overlap, to first-strand cDNA, generating a new 5' end. Subsequent PCR with a down-stream primer and a primer with specificity for this new 5' end leads to products that can easily be cloned and sequenced. Considerations for the choice of primers for ligation and amplification are given. We have used this method to determine the 5' sequences of three independent mRNAs: the human collagen type-X gene, the chicken anchorin CII gene, and the human cytidine deaminase gene. We will discuss this method in comparison with other methods published for the amplification of unknown 5' ends of mRNA species.
我们建立了一种全新的方法来克隆单个cDNA未知长度和序列的5'端。利用T4 DNA连接酶将一对互补引物的退火双链连接到第一链cDNA上，其中一个引物带有4个核苷酸长的随机重叠序列，从而产生一个新的5'端。随后，使用下游引物和针对这个新5'端具有特异性的引物进行PCR，得到的产物可轻松进行克隆和测序。文中给出了连接和扩增引物选择的注意事项。我们已使用该方法确定了三个独立mRNA的5'序列：人类X型胶原基因、鸡锚定蛋白CII基因和人类胞苷脱氨酶基因。我们将把这种方法与已发表的用于扩增mRNA物种未知5'端的其他方法进行比较讨论。
