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20250203004403.0 |
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231222s2001 xx ||||| 00| ||eng c |
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|a pubmed25n0389.xml
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|a (DE-627)NLM116582197
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|a (NLM)11768657
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|a DE-627
|b ger
|c DE-627
|e rakwb
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|a eng
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| 100 |
1 |
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|a Zhang, Y J
|e verfasserin
|4 aut
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| 245 |
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|a Reverse transcription slippage over the mRNA secondary structure of the LIP1 gene
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|c 2001
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|a Text
|b txt
|2 rdacontent
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|a ohne Hilfsmittel zu benutzen
|b n
|2 rdamedia
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|a Band
|b nc
|2 rdacarrier
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| 500 |
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|a Date Completed 30.05.2002
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|a Date Revised 28.09.2018
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|a published: Print
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|a Citation Status MEDLINE
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|a 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
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|a Research Support, Non-U.S. Gov't
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|a Research Support, U.S. Gov't, P.H.S.
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| 650 |
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|a Technical Report
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|a Journal Article
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7 |
|a 5' Untranslated Regions
|2 NLM
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| 650 |
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|a DNA, Complementary
|2 NLM
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| 650 |
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|a Nerve Growth Factors
|2 NLM
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| 650 |
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|a Peptide Fragments
|2 NLM
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| 650 |
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|a RNA, Messenger
|2 NLM
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| 700 |
1 |
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|a Pan, H Y
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Gao, S J
|e verfasserin
|4 aut
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| 773 |
0 |
8 |
|i Enthalten in
|t BioTechniques
|d 1993
|g 31(2001), 6 vom: 29. Dez., Seite 1286, 1288, 1290, passim
|w (DE-627)NLM012627046
|x 0736-6205
|7 nnns
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| 773 |
1 |
8 |
|g volume:31
|g year:2001
|g number:6
|g day:29
|g month:12
|g pages:1286, 1288, 1290, passim
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|d 31
|j 2001
|e 6
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|h 1286, 1288, 1290, passim
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