title: Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution.
abstract: Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization.
authors.affiliation: Guangxi Institute of Animal Sciences, Guangxi Key Laboratory of Livestock Genetic Improvement, Nanning, Guangxi, 530001, P.R. China.
authors.forename: Bingkun
authors.initials: B
authors.lastname: Xie
authors.affiliation: Guangxi Veterinary Research Institute, Nanning, Guangxi, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.affiliation: Hormel Institute, University of Minnesota, Austin, Minnesota, 55912, United States of America.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Ouyang
authors.affiliation: Hormel Institute, University of Minnesota, Austin, Minnesota, 55912, United States of America.
authors.forename: Lichan
authors.initials: L
authors.lastname: Chen
authors.affiliation: College of Animal Science and Technology, Guangxi University, Nanning, 530004, P.R. China.
authors.forename: Hesheng
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Guangxi Institute of Animal Sciences, Guangxi Key Laboratory of Livestock Genetic Improvement, Nanning, Guangxi, 530001, P.R. China.
authors.forename: Yuying
authors.initials: Y
authors.lastname: Liao
authors.affiliation: Department of Pathology, Guizhou Medical University Hospital, Guizhou, Guiyang, 550004, P.R. China.
authors.forename: D Joshua
authors.initials: DJ
authors.lastname: Liao
mesh_terms: D000818:Animals; D016477:Artifacts; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D050939:Gene Fusion / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D012313:RNA / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
chemical_list: D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D012336:RNA, Ribosomal, 16S; D012313:RNA; D004247:DNA