Chenchik A, Diachenko L, Moqadam F, Tarabykin V, Lukyanov S, Siebert P D
CLONTECH Laboratories, Inc., Palo Alto, CA, USA.
Biotechniques. 1996 Sep;21(3):526-34. doi: 10.2144/96213pf02.
An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).
描述了一种高效的cDNA扩增程序，用于确定mRNA的5′和3′末端并克隆全长cDNA。在这种方法中，双链（ds）衔接子通过T4 DNA连接酶连接到ds cDNA文库的两端。然后，使用这种衔接子连接的ds cDNA，通过基因特异性引物和衔接子特异性引物的组合，通过PCR选择性扩增5′或3′ cDNA片段。这是一种从相同的衔接子连接的ds cDNA模板进行5′和3′ cDNA末端快速扩增（RACE）的统一方法。一种专门设计的衔接子结合了“载体PCR”和“抑制PCR”技术的特点，可在扩增过程中显著降低背景。“长片段精确PCR”（LA PCR）技术的应用使得扩增大型RACE产物和全长cDNA成为可能，且对原始mRNA具有高保真度。我们通过扩增人类肌动蛋白基因家族三个成员（1.3 - 1.9 kb）、中等丰度的转铁蛋白受体mRNA（5.0 kb）和低 - 中等丰度的胰岛素样生长因子II受体mRNA（9.1 kb）的5′和3′ RACE片段以及全长cDNA，研究了这种基于PCR的cDNA克隆方法的有效性和局限性。
