title: Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription.
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.
authors.affiliation: Department of Medicine, Center for Functional Genomics, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637, USA.
authors.forename: Douglas Kyung
authors.initials: DK
authors.lastname: Nam
authors.forename: Sanggyu
authors.initials: S
authors.lastname: Lee
authors.forename: Guolin
authors.initials: G
authors.lastname: Zhou
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Cao
authors.forename: Clarence
authors.initials: C
authors.lastname: Wang
authors.forename: Terry
authors.initials: T
authors.lastname: Clark
authors.forename: Jianjun
authors.initials: J
authors.lastname: Chen
authors.forename: Janet D
authors.initials: JD
authors.lastname: Rowley
authors.forename: San Ming
authors.initials: SM
authors.lastname: Wang
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D020224:Expressed Sequence Tags; D006801:Humans; D008957:Models, Genetic; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D011061:Poly A / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D011061:Poly A