Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription.
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.
Nam
Douglas Kyung
DK
Department of Medicine, Center for Functional Genomics, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637, USA.
Lee
Sanggyu
S
Zhou
Guolin
G
Cao
Xiaohong
X
Wang
Clarence
C
Clark
Terry
T
Chen
Jianjun
J
Rowley
Janet D
JD
Wang
San Ming
SM
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
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D011061:Poly A