title: Reverse transcription slippage over the mRNA secondary structure of the LIP1 gene.
abstract: 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.
authors.affiliation: The University of Texas Health Science Center at San Antonio, USA.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Zhang
authors.forename: H Y
authors.initials: HY
authors.lastname: Pan
authors.forename: S J
authors.initials: SJ
authors.lastname: Gao
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D006358:Hot Temperature; D006801:Humans; D008223:Lymphoma; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D020121:5' Untranslated Regions; D018076:DNA, Complementary; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D012333:RNA, Messenger