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):2555-62. doi: 10.1038/nprot.2006.480.
The 5' ends of transcripts provide important information about transcription initiation sites and the approximate locations of local cis-acting enhancer elements; it is therefore important to establish the 5' ends with some precision. RACE (rapid amplification of cDNA ends) PCR is useful for quickly obtaining full length cDNAs for mRNAs for which only part of the sequence is known and to identify alternative 5' or 3' ends of fully sequenced genes. The method consists of using PCR to amplify, from complex mixtures of cellular mRNA, the regions between the known parts of the sequence and non-specific tags appended to the ends of the cDNA. Whereas the poly(A) tail serves to provide such a tag at the 3' end of the mRNA, an artificial one needs to be generated at the 5' end, and various approaches have been described to address this step. The classical scheme for 5' RACE described here is simple, suffices in many instances in which RACE is needed and can be performed in 1-3 days.
转录本的5'端提供了有关转录起始位点和局部顺式作用增强子元件大致位置的重要信息；因此，精确确定5'端很重要。RACE（cDNA末端快速扩增）PCR可用于快速获得仅已知部分序列的mRNA的全长cDNA，并鉴定已完全测序基因的可变5'或3'端。该方法包括使用PCR从细胞mRNA的复杂混合物中扩增序列已知部分与附加到cDNA末端的非特异性标签之间的区域。虽然聚腺苷酸尾在mRNA的3'端提供了这样一个标签，但需要在5'端生成一个人工标签，并且已经描述了各种方法来完成这一步骤。这里描述的经典5'RACE方案很简单，在许多需要RACE的情况下都足够了，并且可以在1-3天内完成。
