Ozawa Tatsuhiko, Kondo Masato, Isobe Masaharu
Laboratory of Molecular and Cellular Biology. Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan.
J Hum Genet. 2004;49(2):102-105. doi: 10.1007/s10038-003-0109-0. Epub 2004 Jan 22.
The 3' rapid amplification of cDNA ends (3' RACE) is widely used to isolate the cDNA of unknown 3' flanking sequences. However, the conventional 3' RACE often fails to amplify cDNA from a large transcript if there is a long distance between the 5' gene-specific primer and poly(A) stretch, since the conventional 3' RACE utilizes 3' oligo-dT-containing primer complementary to the poly(A) tail of mRNA at the first strand cDNA synthesis. To overcome this problem, we have developed an improved 3' RACE method suitable for the isolation of cDNA derived from very large transcripts. By using the oligonucleotide-containing random 9mer together with the GC-rich sequence for the suppression PCR technology at the first strand of cDNA synthesis, we have been able to amplify the cDNA from a very large transcript, such as the microtubule-actin crosslinking factor 1 (MACF1) gene, which codes a transcript of 20 kb in size. When there is no splicing variant, our highly specific amplification allows us to perform the direct sequencing of 3' RACE products without requiring cloning in bacterial hosts. Thus, this stepwise 3' RACE walking will help rapid characterization of the 3' structure of a gene, even when it encodes a very large transcript.
3' cDNA末端快速扩增（3' RACE）被广泛用于分离未知3'侧翼序列的cDNA。然而，如果5'基因特异性引物与poly(A)尾之间距离较长，传统的3' RACE往往无法从大转录本中扩增出cDNA，因为传统3' RACE在第一链cDNA合成时利用与mRNA的poly(A)尾互补的含3'寡聚dT引物。为克服这一问题，我们开发了一种改进的3' RACE方法，适用于分离源自非常大的转录本的cDNA。通过在第一链cDNA合成时将含寡核苷酸的随机9聚体与富含GC的序列一起用于抑制PCR技术，我们能够从非常大的转录本中扩增出cDNA，比如编码大小为20 kb转录本的微管-肌动蛋白交联因子1（MACF1）基因。当不存在剪接变体时，我们高度特异性的扩增使我们能够直接对3' RACE产物进行测序，而无需在细菌宿主中克隆。因此，这种逐步的3' RACE步移将有助于快速鉴定基因的3'结构，即使它编码一个非常大的转录本。
