Zhang Y J, Pan H Y, Gao S J
The University of Texas Health Science Center at San Antonio, USA.
Biotechniques. 2001 Dec;31(6):1286, 1288, 1290, passim. doi: 10.2144/01316st02.
The secondary structures in mRNA often cause early termination during the synthesis of cDNA. In an attempt to determine the 5'-untranslated region (UTR) of the gene LIP1 using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we found that reverse transcriptases skipped over the LIP1 RNA secondary structures and continued the DNA synthesis through RNA adapter sequences without early termination. A fragment of only three nucleotides upstream of the LIP1 translation initiation codon was obtained from the initial RACE-PCR, which was much shorter than the 57-nucleotide fragment obtained from the cDNA library screening. Analysis of the 5' end sequence indicates the presence of high G+C content and stem-loop secondary structures. Therefore, optimizations of the reaction with high temperature (70 degrees C) and a thermostable reverse transcriptase were performed to synthesize the first-strand cDNA, which was determined to have 73 nucleotides in the 5'-UTR. These results suggest that, under cDNA synthesis conditions at 42 degrees C and 60 degrees C, the reverse transcriptases skipped over the stem-loop structures of LIP1 mRNA and continued the cDNA synthesis until they reached the RNA adapter sequences. Thisfinding draws attention to adopting optimized conditions for cDNA synthesis on G+C-rich RNA templates.
mRNA中的二级结构常常会在cDNA合成过程中导致提前终止。在尝试使用RNA连接酶介导的cDNA末端快速扩增（RLM-RACE）来确定LIP1基因的5'-非翻译区（UTR）时，我们发现逆转录酶会跳过LIP1 RNA的二级结构，并通过RNA衔接子序列继续DNA合成而不会提前终止。从最初的RACE-PCR中获得了LIP1翻译起始密码子上游仅三个核苷酸的片段，这比从cDNA文库筛选中获得的57个核苷酸的片段短得多。对5'端序列的分析表明存在高G+C含量和茎环二级结构。因此，进行了高温（70摄氏度）和耐热逆转录酶反应的优化，以合成第一链cDNA，确定其5'-UTR中有73个核苷酸。这些结果表明，在42摄氏度和60摄氏度的cDNA合成条件下，逆转录酶跳过了LIP1 mRNA的茎环结构，并继续进行cDNA合成，直到它们到达RNA衔接子序列。这一发现提醒人们注意在富含G+C的RNA模板上采用优化的cDNA合成条件。
