Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription

Douglas Kyung Nam, Sanggyu Lee, Guolin Zhou, Xiaohong Cao, Clarence Wang, Terry Clark, Jianjun Chen, Janet D. Rowley, San Ming Wang

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Abstract

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

Original languageEnglish (US)
Pages (from-to)6152-6156
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number9
DOIs
StatePublished - Apr 30 2002

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