Scotto-Lavino Elizabeth, Du Guangwei, Frohman Michael A
Graduate Program in Molecular & Cellular Pharmacology, Stony Brook University, Stony Brook, New York 11794, USA.
Nat Protoc. 2006;1(6):2742-5. doi: 10.1038/nprot.2006.481.
Having knowledge of the entire 3' sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA. Also, some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties, or change the encoded protein. Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE) PCR as long as part of the mRNA sequence is known; adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified. In 3' RACE, the poly(A) tail functions as a non-specific tag at the 3' end of the mRNA. cDNA ends can be obtained in 1-3 days using this protocol.
了解cDNA的整个3'序列通常很重要，因为非编码末端区域可能包含调节mRNA稳定性或亚细胞定位的信号。此外，一些信使RNA使用替代基因组位点进行切割和聚腺苷酸化，这可能会改变上述特性，或改变编码的蛋白质。只要知道部分mRNA序列，就可以使用cDNA末端快速扩增（RACE）PCR从细胞mRNA的复杂混合物中获得全长cDNA；在cDNA末端添加非特异性标签可以扩增序列已知部分与末端之间的区域。在3'RACE中，聚（A）尾在mRNA的3'末端起非特异性标签的作用。使用本方案可在1-3天内获得cDNA末端。
